Structure of recombinant human renin, a target for cardiovascular-active drugs, at 2.5 A resolution.

The x-ray crystal structure of recombinant human renin has been determined. Molecular dynamics techniques that included crystallographic data as a restraint were used to improve an initial model based on porcine pepsinogen. The present agreement factor for data from 8.0 to 2.5 angstroms (A) is 0.236. Some of the surface loops are poorly determined, and these disordered regions border a 30 A wide solvent channel. Comparison of renin with other aspartyl proteinases shows that, although the structural cores and active sites are highly conserved, surface residues, some of which are critical for specificity, vary greatly (up to 10A). Knowledge of the actual structure, as opposed to the use of models based on related enzymes, should facilitate the design of renin inhibitors.

Alpha 1 adrenergic receptor-induced c-fos gene expression in rat aorta and cultured vascular smooth muscle cells.

While growth of blood vessels is important in hypertension, relatively little is known about the contribution of catecholamines. Using isolated rat aorta and cultured smooth muscle cells, we examined adrenergic stimulation of gene expression. Phenylephrine, a selective alpha 1 adrenergic receptor agonist, caused a rapid and transient increase in c-fos mRNA accumulation which was inhibited by prazosin, an alpha 1 receptor antagonist. Similarly, phenylephrine stimulated c-jun and c-myc mRNA accumulation. Chloroethyl-clonidine, a compound which irreversibly blocks alpha 1B receptors, completely blocked the phenylephrine-induced increase in c-fos mRNA. RNase protection experiments demonstrated that rat aorta prominently expressed mRNA for alpha 1B and alpha 1A/D receptors. Phenylephrine-induced c-fos mRNA was partially inhibited by H-7, a protein kinase C inhibitor, and by nifedipine, a Ca2+ channel blocker; these two compounds together had additive effects. In situ hybridization showed that expression of c-fos mRNA induced by phenylephrine was localized to aorta's medial layer. These results suggest that alpha 1 receptor-induced increase in c-fos mRNA in aorta is mediated by a chloroethyl-clonidine-sensitive receptor subtype signaling via increasing intracellular Ca2+ concentrations and activating protein kinase C.

Cloning of the mouse gene for D-dopachrome tautomerase.

D-Dopachrome tautomerase converts 2-carboxy-2,3-dihydroindole-5, 6-quinone (D-dopachrome) into 5,6-dihydroxyindole. The amino acid sequence of this protein is 27% identical with that of macrophage migration inhibitory factor, which is known as a cytokine, pituitary hormone, and glucocorticoid-induced immunomodulator. In this study, we isolated and sequenced a 3490 bp-long genomic DNA of mouse D-dopachrome tautomerase that consists of three exons and two introns. By two procedures, 5' rapid amplification of cDNA ends and cap site labeling, we determined the transcription initiation site, which is located 46 bp upstream of the translation initiation site. The possible polyadenylation sequence (AATAAA) is located 180 bp downstream of the termination codon. Computer-assisted analysis of the nucleotide sequence revealed a number of regulatory motifs, including multiple sites for Sp1, C/EBP, NF-Y, and USF. Although the precise pathophysiological functions of D-dopachrome tautomerase remain to be elucidated, the present results will contribute not only to elucidation of the mechanism of gene expression, but also to understanding of the molecular function of this protein.

Rat submaxillary gland serine protease, tonin. Structure solution and refinement at 1.8 A resolution.

Tonin is a mammalian serine protease that is capable of generating the vasoconstrictive agent, angiotensin II, directly from its precursor protein, angiotensinogen, a process that normally requires two enzymes, renin and angiotensin-converting enzyme. The X-ray crystallographic structure determination and refinement of tonin at 1.8 A resolution and the analysis of the resulting model are reported. The initial phases were obtained by the method of molecular replacement using as the search model the structure of bovine trypsin. The refined model of tonin consists of 227 amino acid residues out of the 235 in the complete molecule, 149 water molecules, and one zinc ion. The R-factor (R = sigma Fo - Fc/sigma Fo) is 0.196 for the 14,997 measured data between 8 and 1.8 A resolution with I greater than or equal to sigma (I). It is estimated that the overall root-mean-square error in the coordinates is about 0.3 A. The structure of tonin that has been determined is not in its active conformation, but one that has been perturbed by the binding of Zn2+ in the active site. Zn2+ was included in the buffer to aid the crystallization. Nevertheless, the structure of tonin that is described is for the most part similar to its native form as indicated by the close tertiary structural homology with kallikrein. The differences in the structures of the two enzymes are concentrated in several loop regions; these structural differences are probably responsible for the differences in their reactivities and specificities.

Refined structure of porcine pepsinogen at 1.8 A resolution.

The molecular structure of porcine pepsinogen at 1.8 A resolution has been determined by a combination of molecular replacement and multiple isomorphous phasing techniques. The resulting structure was refined by restrained-parameter least-squares methods. The final R factor [formula: see text] is 0.164 for 32,264 reflections with I greater than or equal to sigma (I) in the resolution range of 8.0 to 1.8 A. The model consists of 2785 protein atoms in 370 residues, a phosphoryl group on Ser68 and 238 ordered water molecules. The resulting molecular stereochemistry is consistent with a well-refined crystal structure with co-ordinate accuracy in the range of 0.10 to 0.15 A for the well-ordered regions of the molecule (B less than 15 A2). For the enzyme portion of the zymogen, the root-mean-square difference in C alpha atom co-ordinates with the refined porcine pepsin structure is 0.90 A (284 common atoms) and with the C alpha atoms of penicillopepsin it is 1.63 A (275 common atoms). The additional 44 N-terminal amino acids of the prosegment (Leu1p to Leu44p, using the letter p after the residue number to distinguish the residues of the prosegment) adopt a relatively compact structure consisting of a long beta-strand followed by two approximately orthogonal alpha-helices and a short 3(10)-helix. Intimate contacts, both electrostatic and hydrophobic interactions, are made with residues in the pepsin active site. The N-terminal beta-strand, Leu1p to Leu6p, forms part of the six-stranded beta-sheet common to the aspartic proteinases. In the zymogen the first 13 residues of pepsin, Ile1 to Glu13, adopt a completely different conformation from that of the mature enzyme. The C alpha atom of Ile1 must move approximately 44 A in going from its position in the inactive zymogen to its observed position in active pepsin. Electrostatic interactions of Lys36pN and hydrogen-bonding interactions of Tyr37pOH, and Tyr90H with the two catalytic aspartate groups, Asp32 and Asp215, prevent substrate access to the active site of the zymogen. We have made a detailed comparison of the mammalian pepsinogen fold with the fungal aspartic proteinase fold of penicillopepsin, used for the molecular replacement solution. A structurally derived alignment of the two sequences is presented.

Refined structure of alpha-lytic protease at 1.7 A resolution. Analysis of hydrogen bonding and solvent structure.

The structure of alpha-lytic protease, a serine protease produced by the bacterium Lysobacter enzymogenes, has been refined at 1.7 A resolution. The conventional R-factor is 0.131 for the 14,996 reflections between 8 and 1.7 A resolution with I greater than or equal to 2 sigma (I). The model consists of 1391 protein atoms, two sulfate ions and 156 water molecules. The overall root-meansquare error is estimated to be about 0.14 A. The refined structure was compared with homologous enzymes alpha-chymotrypsin and Streptomyces griseus protease A and B. A new sequence numbering was derived based on the alignment of these structures. The comparison showed that the greatest structural homology is around the active site residues Asp102, His57 and Ser195, and that basic folding pathways are maintained despite chemical changes in the hydrophobic cores. The hydrogen bonds in the structure were tabulated and the distances and angles of interaction are similar to those found in small molecules. The analysis also revealed the presence of close intraresidue interactions. There are only a few direct intermolecular hydrogen bonds. Most intermolecular interactions involve bridging solvent molecules. The structural importance of hydrogen bonds involving the side-chain of Asx residues is discussed. All the negatively charged groups have a counterion nearby, while the excess positively charged groups are exposed to the solvent. One of the sulfate ions is located near the active site, whereas the other is close to the N terminus. Of the 156 water molecules, only seven are not involved in a hydrogen bond. Six of these have polar groups nearby, while the remaining one is in very weak density. There are nine internal water molecules, consisting of two monomers, two dimers and one trimer. No significant second shell of solvent is observed.

Computational analysis of the binding of P1 variants of domain 3 of turkey ovomucoid inhibitor to Streptomyces griseus protease B.

Binding constants for complexes of variants of the ovomucoid inhibitor domain 3 from turkey (OMTKY3) and Streptomyces griseus protease B (SGPB) have been computed. On the basis of the crystallographically determined structures of the complexes, continuum electrostatic calculations have been carried out to evaluate the electrostatic contribution to the binding energy. The hydrophobic component was computed based on the change in the solvent accessible surface area on complex formation. These two terms were combined linearly and the parameters for the protein dielectric, atomic solvation parameter and a constant term were derived using a multivariate fit to the observed binding energies. The resulting fit shows a high correlation with a multiple coefficient of determination of 0.79. This indicates that 79% of the variation in the observed binding energies is explained by the electrostatic and hydrophobic terms. The analysis results in a protein dielectric of 8.2 and an atomic solvation parameter of 30 cal/mol A2. As a test, these parameters were used to calculate the binding energies of complexes of chymotrypsin and of leukocyte elastase OMTKY3, as well as three other variants of OMTKY3 bound to SGPB. As these structures were not used for the multivariate fit, they serve as an independent check on the derived parameters. The calculated energies for the three new variants of OMTKY3 are in good agreement with the observed values. However, the binding energies of the other complexes are poorly predicted. This implies that the parameters that were obtained are not transferable. The possible causes for this lack of transferability are discussed.

Refined crystal structure of the seryl-tRNA synthetase from Thermus thermophilus at 2.5 A resolution.

The three-dimensional structure of the seryl-tRNA synthetase from Thermus thermophilus has been determined and refined at 2.5 A resolution. The final model consists of a dimer of 421 residues each and 190 water molecules. The R-factor is 18.4% for all the data between 10 and 2.5 A resolution. The structure is very similar to that of the homologous enzyme from Escherichia coli, with an r.m.s. difference of 1.5 A for the 357 alpha-carbon atoms considered equivalent. The comparison of the two structures indicates increased hydrophobicity, reduced conformational entropy and reduced torsional strain as possible mechanisms by which thermostability is obtained in the enzyme from the thermophile.

Crystal and molecular structures of the complex of alpha-chymotrypsin with its inhibitor turkey ovomucoid third domain at 1.8 A resolution.

The molecular structure of the complex between bovine pancreatic alpha-chymotrypsin (EC 3.4.4.5) and the third domain of the Kazal-type ovomucoid from Turkey (OMTKY3) has been determined crystallographically by the molecular replacement method. Restrained-parameter least-squares refinement of the molecular model of the complex has led to a conventional agreement factor R of 0.168 for the 19,466 reflections in the 1.8 A (1 A = 0.1 nm) resolution shell [I greater than or equal to sigma (I)]. The reactive site loop of OMTKY3, from Lys13I to Arg21I (I indicates inhibitor), is highly complementary to the surface of alpha-chymotrypsin in the complex. A total of 13 residues on the inhibitor make 113 contacts of less than 4.0 A with 21 residues of the enzyme. A short contact (2.95 A) from O gamma of Ser195 to the carbonyl-carbon atom of the scissile bond between Leu18I and Glu19I is present; in spite of it, this peptide remains planar and undistorted. Analysis of the interactions of the inhibitor with chymotrypsin explains the enhanced specificity that chymotrypsin has for P'3 arginine residues. There is a water-mediated ion pair between the guanidinium group on this residue and the carboxylate of Asp64. Comparison of the structure of the alpha-chymotrypsin portion of this complex with the several structures of alpha and gamma-chymotrypsin in the uncomplexed form shows a high degree of structural equivalence (root-mean-square deviation of the 234 common alpha-carbon atoms averages 0.38 A). Significant differences occur mainly in two regions Lys36 to Phe39 and Ser75 to Lys79. Among the 21 residues that are in contact with the ovomucoid domain, only Phe39 and Tyr146 change their conformations significantly as a result of forming the complex. Comparison of the structure of the OMTKY3 domain in this complex to that of the same inhibitor bound to a serine proteinase from Streptomyces griseus (SGPB) shows a central core of 44 amino acids (the central alpha-helix and flanking small 3-stranded beta-sheet) that have alpha-carbon atoms fitting to within 1.0 A (root-mean-square deviation of 0.45 A) whereas the residues of the reactive-site loop differ in position by up to 1.9 A (C alpha of Leu18I). The ovomucoid domain has a built-in conformational flexibility that allows it to adapt to the active sites of different enzymes. A comparison of the SGPB and alpha-chymotrypsin molecules is made and the water molecules bound at the inhibitor-enzyme interface in both complexes are analysed for similarities and differences.

The crystal structure of PR3, a neutrophil serine proteinase antigen of Wegener's granulomatosis antibodies.

The crystal structure of PR3, a serine proteinase from the azurophilic granules of human polymorphonuclear neutrophils, has been solved by molecular replacement using the human leukocyte elastase structure. The PR3 structure has been refined to an R-factor (= sigma parallel Fo magnitude of-Fc parallel/sigma magnitude of Fo) of 0.201 for all data in the range of 10.0 to 2.2 A resolution. The enzyme was crystallized in space group P21 with four molecules in the asymmetric unit (Vm approximately equal to 2.6 A/Da). The overall fold consists of two domains of beta-barrel structures typical of the chymotrypsin family of serine proteinases. In general, the substrate binding sites, S4 to S3', are more polar than comparable sites in the related proteinase, human leukocyte elastase. The experimentally observed preference of PR3 for small aliphatic residues at the P1 position of a substrate is explained by the Val to Ile substitution at position 190 when compared to the elastase structure. The substitution of Ala by Asp at position 213 at the back of S1 should not affect its specificity greatly, as the Asp side-chain points back into the interior of the protein. The PR3 structure includes a disaccharide unit (N-linked 2-acetamido-2-deoxy-beta-D-glucopyranose and 1,6-linked alpha-L-fucopyranose) covalently attached to Asn 159. The linear antigenic sites of PR3 reported to react with Wegener's granulomatosis autoantibodies occur in regions of the three-dimensional structure that may implicate the inactive pro-form of the enzyme in the pathogenesis of the disease.

Modulation of vascular development and injury by angiotensin II.

OBJECTIVE: To examine the exact profile of expression and to determine the functional significance of the angiotensin II (Ang II), type I (AT1) and type 2 (AT2) receptors during rat aortic development and following rat carotid artery balloon injury. METHODS: AT1 and AT2 mRNA levels in rat aortae were measured using a quantitative reverse transcription polymerase chain reaction technique. Ang II receptor function was assessed by quantitating the effects of AT1 (DuP753) and AT2 (PD123319) receptor antagonists during these processes. RESULTS: During aortic development, AT1 expression was detected on gestational day 14, increased until embryonic day 16 (E16), after which, levels were similar throughout postnatal development. Conversely, AT2 mRNA first appeared at E16, reached maximal levels between E19 and neonatal day 1, and decreased thereafter. DNA synthesis rates decreased with aortic development (high at E15, 73.8 +/- 3.1%; dropping to 37.5 +/- 2.3% by E21). Whereas AT1 receptor antagonism accelerated this developmentally regulated decrease in DNA synthesis. AT2 receptor antagonism blunted this decrease. Because activated adult medial smooth muscle cells express a neonatal phenotype after vascular injury, we assessed Ang II receptor levels and function after carotid artery balloon injury. Both receptor subtypes increased; however, AT2 receptor mRNA expression peaked earlier than AT1 (48 to 72 h after injury). As with aortic development, DNA synthesis occurring between 24 to 48 h after injury (when AT2 receptors constitute 10% of the Ang II receptor population) decreased in DuP753-treated animals and increased in PD123319-treated animals. CONCLUSION: These results indicate that Ang II receptors play a role in vascular development by promoting opposing effects on vascular smooth muscle cell growth.

Crystal structure of human pepsin and its complex with pepstatin.

The three-dimensional crystal structure of human pepsin and that of its complex with pepstatin have been solved by X-ray crystallographic methods. The native pepsin structure has been refined with data collected to 2.2 A resolution to an R-factor of 19.7%. The pepsin:pepstatin structure has been refined with data to 2.0 A resolution to an R-factor of 18.5%. The hydrogen bonding interactions and the conformation adopted by pepstatin are very similar to those found in complexes of pepstatin with other aspartic proteinases. The enzyme undergoes a conformational change upon inhibitor binding to enclose the inhibitor more tightly. The analysis of the binding sites indicates that they form an extended tube without distinct binding pockets. By comparing the residues on the binding surface with those of the other human aspartic proteinases, it has been possible to rationalize some of the experimental data concerning the different specificities. At the S1 site, valine at position 120 in renin instead of isoleucine, as in the other enzymes, allows for binding of larger hydrophobic residues. The possibility of multiple conformations for the P2 residue makes the analysis of the S2 site difficult. However, it is possible to see that the specific interactions that renin makes with histidine at P2 would not be possible in the case of the other enzymes. At the S3 site, the smaller volume that is accessible in pepsin compared to the other enzymes is consistent with its preference for smaller residues at the P3 position.

Expression of alpha 1 adrenergic receptor subtype mRNAs in the rat cardiovascular system with aging.

Alpha 1 adrenoreceptors mediate inotropic and chronotropic responses in the heart and vaso-constriction in blood vessels. Pharmacological studies using selective antagonists for subtypes of alpha 1 receptor suggest that different receptor subtypes may mediate different physiological effects. Prior studies have demonstrated that alpha 1-mediated cardiac responses are altered in aging and that alpha 1 receptor number declines with age in the rat heart. The distribution of alpha 1 receptor subtypes in cardiac tissue and subtype-specific changes with aging, however, have not been established. Using R Nase protection assays and in situ hybridization techniques, we have detected message for alpha 1B, alpha 1A, and alpha 1D in all four chambers of the rat heart and in multiple blood vessels. We found no significant changes in alpha 1 receptor subtype mRNA levels in hearts from young (4 months) and old (25 months) Sprague-Dawley rats. Also, there was expression of these three subtypes in all blood vessels assayed. We conclude that transcriptional regulation of alpha 1 adrenoreceptor subtypes does not account for age-related changes in cardiac alpha-adrenergic responsiveness.

Opioidergic and adrenergic modulation of formalin-evoked spinal c-fos mRNA expression and nocifensive behavior in the rat.

Fos protein expression has been used to reflect neuronal activation in pain processing pathways although analgesics may uncouple behavioral and Fos responses. We determine whether formalin-induced spinal c-fos mRNA expression (Northern blotting) correlates with nocifensive behavior following pretreatment with morphine, the alpha2-adrenoceptor agonist dexmedetomidine, or their respective antagonists naloxone and atipamezole. Both opiate and alpha2-adrenoceptor agonists reduced formalin-induced c-fos gene transcription and nocifensive behavior via their cognate receptors. Unexpectedly, blockade of either the opiate or alpha2-adrenergic receptors, alone, caused an increase in formalin-evoked c-fos mRNA; while blocking the opiate receptor had no effect on formalin-induced behavior, alpha2-adrenoceptor block had an analgesic effect, indicating discordance between c-fos message transcription and nocifensive behavior. We concluded that the formalin-induced spinal c-fos signal was a poor predictor of the behavioral response to pharmacological manipulation of pain processing pathways.

Gene expression of catecholamine synthesizing enzymes and beta adrenoceptor subtypes during rat embryogenesis.

Timed-pregnant Sprague-Dawley rats were killed between gestational day (GD) 8 and 10, and embryos were explanted and separated into developmental stages according to a modified Theiler's system. Total RNA from each stage was isolated and subjected to reverse transcription-polymerase chain reaction (RT-PCR) assays to examine gene expression of catecholamine synthesizing enzymes and three subtypes of beta adrenoceptors. Expression of these genes was detected at much earlier stages than previously reported, and each enzyme and receptor subtype showed a different pattern of gene expression. For example, mRNA for tyrosine hydroxylase, the rate-limiting enzyme for catecholamine synthesis, was detected as early as stage 10a, late GD 8, before the neural crest cells appear (stage 12, mid GD 10). This contradicts the common belief that catecholamines are produced only in the cells of sympathoadrenal lineage which originate from the neural crest cells and the cells of central nervous system. Results from the present study indicate that catecholamine synthesis is not limited to the cells of sympathoadrenal lineage.

Remission of a recurrent carpal tunnel syndrome by a new device of the hemodialysis method in a long-term hemodialysis patient.

This report concerns a case in which remission was achieved from the recurrent carpal tunnel syndrome employing new methods of hemodialysis. These being the maintenance of low endotoxin in dialysate, a highly permeable membrane and a 32-microglobulin-adsorbent column. A 78-year-old female patient with a 19-year history of hemodialysis was diagnosed as being a suitable recipient of a third operation. The concentration of endotoxin was maintained at under 10 EU/l and the highly permeable dialyzer with a larger sieving coefficient of beta2-microglobulin was introduced. A Lixelle adsorption column for beta2-microglobulin removal was also introduced and the serum concentration of the beta-microglobulin was maintained at under 20 mg/dl. Consequently, within 6 months the symptoms in the right hand had completely disappeared, the motor nerve latency had almost normalized at 5.0 msec and no recurrence was observed.

Rat whole embryo culture system as a tool to investigate developmental mechanisms of left/right body axis.

The rat whole embryo culture (WEC) system is a suitable experimental tool for the study of the mechanisms of development of the left/right body axis (L/R axis) because embryos can be cultured from before and during the development of several asymmetric structures including the heart. This paper reviews the development of asymmetric structures in rat embryos during the culture period and the literature on abnormal development of the L/R axis studied using the WEC system. In addition, the author suggests the following guideline for investigators who use this system to study the development of the L/R axis. (1) Precisely stage the embryos according to a modified Theiler's system. (2) Examine the sidedness of three asymmetric structures whose sidedness is controlled by different mechanisms [i.e. the heart, chorioallantoic placenta and the lower part of the embryo (the so-called 'tail')]. (3) Establish the background incidences of inversion of the above structures in control groups cultured from different stages. (4) Perform dose-response studies if testing a chemical. (5) Examine antagonists or inactive isoforms to confirm the pharmacological effects of the test chemical.

Expression of mRNA for alpha1-adrenoceptor subtypes at the beginning of neurulation in rat embryos.

We recently reported that alpha1-adrenoceptor agonists, administered at the beginning of neurulation (Stage 11a) in rat embryos grown in culture, interfere with normal development of the left/right body axis leading to situs inversus. Despite these pharmacological findings, expression of alpha1-adrenoceptor genes at such an early stage of development has not been demonstrated. In the present study, we examined the expression of mRNAs for cloned alpha1-adrenoceptor subtypes in rat embryos at Stage 11a. Timed-pregnant Sprague-Dawley rats were killed in the morning of gestational day 9 (vaginal plug day = day 0), and the implantation sites were removed. The implantation sites were separated into embryo, ectoplacental cone and decidua, only those at Stage 11a were selected, and these were immediately frozen on dry ice, and subsequently their total RNA was isolated. RNase protection assays were then performed for cloned alpha1a-, alpha 1b- and alpha1d-adrenocepter subtypes using 20-30 mug of total RNA for each hybridization. In all tissues, strong and weak signals were detected for alpha1b- and alpha1a-adrenoceptor subtype mRNAs, respectively. In contrast, a signal for alpha1d mRNA was not detected in any tissues. These results, together with previous pharmacological findings, suggest the existence of alpha1a- and alpha1b-adrenoceptor subtypes in rat embryos at Stage 11a.

Characterization of the rat adrenal medulla cultured in vitro.

A wide variety of experimental animal models have been used to investigate the mechanisms of synthesis, storage, and release of catecholamines. Whereas in vivo experimental models are situated at one end of the spectrum, cell culture models are situated at the other end. In the present study, we have characterized various aspects of the rat adrenal medulla cultured in vitro as a whole tissue, aiming to establish a new experimental model in between in vivo animal models and cell culture models. We adapted a bottle rotator system commonly used for culturing rodent whole embryos. Changes in histology, activities and mRNA levels of catecholamine-synthesizing enzymes, and concentrations of catecholamines in the adrenal medulla were studied. In addition, the effects of cholinergic stimulation on catecholamine release from the adrenal medulla were examined. Overall the results indicate that various aspects of the adrenal medulla become stable after 4 d of culture and the adrenal medulla at this stage releases catecholamines in response to cholinergic stimulation. The whole adrenal medulla culture system may be a useful tool for investigating catecholamine-related functions dependent on intercellular reactions or communications.