Structure determination of the extracellular xylanase from Geobacillus stearothermophilus by selenomethionyl MAD phasing.

Xylanases are hemicellulases that hydrolyze the internal beta-1,4-glycoside bonds of xylan. The extracellular thermostable endo-1,4-beta-xylanase (EC 3.2.1.8; XT6) produced by the thermophilic bacterium Geobacillus stearothermophilus T-6 was shown to bleach pulp optimally at pH 9 and 338 K and was successfully used in a large-scale biobleaching mill trial. The xylanase gene was cloned and sequenced. The mature enzyme consists of 379 amino acids, with a calculated molecular weight of 43 808 Da and a pI of 9.0. Crystallographic studies of XT6 were performed in order to study the mechanism of catalysis and to provide a structural basis for the rational introduction of enhanced thermostability by site-specific mutagenesis. XT6 was crystallized in the primitive trigonal space group P3(2)21, with unit-cell parameters a = b = 112.9, c = 122.7 A. A full diffraction data set for wild-type XT6 has been measured to 2.4 A resolution on flash-frozen crystals using synchrotron radiation. A fully exchanged selenomethionyl XT6 derivative (containing eight Se atoms per XT6 molecule) was also prepared and crystallized in an isomorphous crystal form, providing full selenium MAD data at three wavelengths and enabling phase solution and structure determination. The structure of wild-type XT6 was refined at 2.4 A resolution to a final R factor of 15.6% and an R(free) of 18.6%. The structure demonstrates that XT6 is made up of an eightfold TIM-barrel containing a deep active-site groove, consistent with its 'endo' mode of action. The two essential catalytic carboxylic residues (Glu159 and Glu265) are located at the active site within 5.5 A of each other, as expected for 'retaining' glycoside hydrolases. A unique subdomain was identified in the carboxy-terminal part of the enzyme and was suggested to have a role in xylan binding. The three-dimensional structure of XT6 is of great interest since it provides a favourable starting point for the rational improvement of its already high thermal and pH stabilities, which are required for a number of biotechnological and industrial applications.

Structural basis for selective recognition of oligosaccharides by DC-SIGN and DC-SIGNR.

Dendritic cell specific intracellular adhesion molecule-3 (ICAM-3) grabbing nonintegrin (DC-SIGN), a C-type lectin present on the surface of dendritic cells, mediates the initial interaction of dendritic cells with T cells by binding to ICAM-3. DC-SIGN and DC-SIGNR, a related receptor found on the endothelium of liver sinusoids, placental capillaries, and lymph nodes, bind to oligosaccharides that are present on the envelope of human immunodeficiency virus (HIV), an interaction that strongly promotes viral infection of T cells. Crystal structures of carbohydrate-recognition domains of DC-SIGN and of DC-SIGNR bound to oligosaccharide, in combination with binding studies, reveal that these receptors selectively recognize endogenous high-mannose oligosaccharides and may represent a new avenue for developing HIV prophylactics.

Mechanism of pH-dependent N-acetylgalactosamine binding by a functional mimic of the hepatocyte asialoglycoprotein receptor.

Efficient release of ligands from the Ca(2+)-dependent carbohydrate-recognition domain (CRD) of the hepatic asialoglycoprotein receptor at endosomal pH requires a small set of conserved amino acids that includes a critical histidine residue. When these residues are incorporated at corresponding positions in an homologous galactose-binding derivative of serum mannose-binding protein, the pH dependence of ligand binding becomes more like that of the receptor. The modified CRD displays 40-fold preferential binding to N-acetylgalactosamine compared with galactose, making it a good functional mimic of the asialoglycoprotein receptor. In the crystal structure of the modified CRD bound to N-acetylgalactosamine, the histidine (His(202)) contacts the 2-acetamido methyl group and also participates in a network of interactions involving Asp(212), Arg(216), and Tyr(218) that positions a water molecule in a hydrogen bond with the sugar amide group. These interactions appear to produce the preference for N-acetylgalactosamine over galactose and are also likely to influence the pK(a) of His(202). Protonation of His(202) would disrupt its interaction with an asparagine that serves as a ligand for Ca(2+) and sugar. The structure of the modified CRD without sugar displays several different conformations that may represent structures of intermediates in the release of Ca(2+) and sugar ligands caused by protonation of His(202).

Structure of a C-type carbohydrate recognition domain from the macrophage mannose receptor.

The mannose receptor of macrophages and liver endothelium mediates clearance of pathogenic organisms and potentially harmful glycoconjugates. The extracellular portion of the receptor includes eight C-type carbohydrate recognition domains (CRDs), of which one, CRD-4, shows detectable binding to monosaccharide ligands. We have determined the crystal structure of CRD-4. Although the basic C-type lectin fold is preserved, a loop extends away from the core of the domain to form a domain-swapped dimer in the crystal. Of the two Ca(2+) sites, only the principal site known to mediate carbohydrate binding in other C-type lectins is occupied. This site is altered in a way that makes sugar binding impossible in the mode observed in other C-type lectins. The structure is likely to represent an endosomal form of the domain formed when Ca(2+) is lost from the auxiliary calcium site. The structure suggests a mechanism for endosomal ligand release in which the auxiliary calcium site serves as a pH sensor. Acid pH-induced removal of this Ca(2+) results in conformational rearrangements of the receptor, rendering it unable to bind carbohydrate ligands.

Conformational changes in E. coli DNA topoisomerase I.

DNA topoisomerases are the enzymes responsible for maintaining the topological states of DNA. In order to change the topology of DNA, topoisomerases pass one or two DNA strands through transient single or double strand breaks in the DNA phosphodiester backbone. It has been proposed that both type IA and type II enzymes change conformation dramatically during the reaction cycle in order to accomplish these transformations. In the case of Escherichia coli DNA topoisomerase I, it has been suggested that a 30 kDa fragment moves away from the rest of the protein to create an entrance into the central hole in the protein. Structures of the 30 kDa fragment reveal that indeed this fragment can change conformation significantly. The fragment is composed of two domains, and while the domains themselves remain largely unchanged, their relative arrangement can change dramatically.

Protein-nucleotide interactions in E. coli DNA topoisomerase I.

DNA topoisomerases are the enzymes responsible for controlling and maintaining the topological states of DNA. Type IA enzymes work by transiently breaking the phosphodiester backbone of one strand to allow passage of another strand through the break. The protein has to perform complex rearrangements of the DNA, and hence it is likely that different regions of the enzyme bind DNA with different affinities. In order to identify some of the DNA binding sites in the protein, we have solved the structures of several complexes of the 67 kDa N-terminal fragment of Escherichia coli DNA topoisomerase I with mono- and trinucleotides. There are five different binding sites in the complexes, one of which is adjacent to the active site. Two other sites are in the central hole of the protein and may represent general DNA binding regions. The positions of these sites allow us to identify different DNA binding regions and to understand their possible roles in the catalytic cycle.

Carboxypeptidase A: native, zinc-removed and mercury-replaced forms.

The crystal structure of the zinc-containing exopeptidase bovine carboxypeptidase A (CPA) has been refined to high resolution, based on a data set collected from a single crystal, incorporating new sequence information based on cloning of the bovine gene. In addition, new refined structures are available for the zinc-removed form of the enzyme, apo-CPA, as well as the mercury-replaced form, Hg-CPA. The native structure reveals that the zinc-bound water molecule does not appear to more loosely bound than the rest of the zinc ligands, at least when B-factor values are considered. Nor is there any evidence for a secondary location of this water molecule. The apo-enzyme structure does not show any density in the place of the removed zinc ion. The only significant change appears to be a chi2 rotation of one zinc histidine ligand to form an ion-pair interaction with a glutamic acid side chain. The structure of Hg-CPA reveals a solvent Tris molecule bound to the mercury cation, as well as an unidentified cation bound to Glu270. The location of this citation agrees with previous proposals for the binding side of inhibitory zinc. These observations may explain some of the differences in kinetics observed in metal- replaced CPA.

Controlled reperfusion after lung ischemia: implications for improved function after lung transplantation.

OBJECTIVES: Despite improvements in organ preservation, reperfusion injury remains a major source of morbidity and mortality after lung transplantation. This pilot study was designed to investigate the effects of controlled reperfusion after lung ischemia. METHODS: Twenty adult pigs underwent 2 hours of warm lung ischemia by crossclamping the left bronchus and pulmonary artery. In five (group 1), the clamp was simply removed at the end of ischemia (uncontrolled reperfusion). The 15 other pigs underwent modified reperfusion using blood from the femoral artery to perfuse the lung through the pulmonary artery (pressure 40 to 50 mm Hg) for 10 minutes before removing the pulmonary artery clamp. In five (group 2), the blood was mixed with crystalloid, resulting in a substrate-enriched, hypocalcemic, hyperosmolar, alkaline solution. In five (group 3), the blood was circulated through a leukocyte-depleting filter, and the last five (group 4) underwent reperfusion with both a modified solution and white blood cell filter. Lung function was assessed 60 minutes after reperfusion, and biopsy specimens were taken. RESULTS: Controlled reperfusion with both a white blood cell filter and modified solution (group 4) completely eliminated the reperfusion injury that occurred with uncontrolled reperfusion (group 1), resulting in complete preservation of compliance (98% +/- 1% vs 77% +/- 1%; p < 0.001, and arterial/alveolar ratio (97% +/- 2% vs 27% +/- 2%; p < 0.001); no increase in pulmonary vascular resistance (106% +/- 1% vs 198% +/- 1%; p < 0.001); lowered tissue edema (82.1% +/- 0.4% vs 84.3% +/- 0.2%; p < 0.001), and myeloperoxidase activity (0.18 +/- 0.02 vs 0.35 +/- 0.02 deltaOD/min/mg protein; p < 0.001). In contrast, using either a white blood cell filter or modified solution separately improved but did not avoid the reperfusion injury, resulting in pulmonary function and tissue edema levels that were intermediate between group 1 (uncontrolled reperfusion) and group 4 (white blood cell filter and modified solution). CONCLUSION: After 2 hours of warm pulmonary ischemia, (1) a severe lung injury occurs after uncontrolled reperfusion, (2) controlled reperfusion with either a modified reperfusion solution or white blood cell filter limits, but does not avoid, a lung reperfusion injury, (3) reperfusion using both a modified reperfusate and white blood cell filter results in complete preservation of pulmonary function. We therefore believe surgeons should control the reperfusate after lung transplantation to improve postoperative pulmonary function.

Detrimental effects of cardiopulmonary bypass in cyanotic infants: preventing the reoxygenation injury.

BACKGROUND: Recent experimental studies have shown that acute hypoxia followed by abrupt reoxygenation using cardiopulmonary bypass (CPB) results in an unintended injury mediated by oxygen free radicals, which can be modified by initiating CPB at a lower fraction of inspired oxygen (FiO2) or by leukocyte filtration. However, the clinical relevance of these experimental studies has been questioned because chronic hypoxia may allow compensatory changes to occur. METHODS: Seven acyanotic infants had CPB initiated at an FiO2 of 1.0. Of 21 cyanotic infants, 7 (group 1) had CPB initiated at an FiO2 of 1.0, 6 (group 2) at an FiO2 of 0.21, and 8 (group 3) underwent CPB using leukocyte filtration. Biopsy of right atrial tissue was performed before and 10 to 20 minutes after the initiation of CPB. The tissue was incubated in 4-mmol/L t-butylhydroperoxide (a strong oxidant), and the malondialdehyde (MDA) level was measured to determine the antioxidant reserve capacity. The more MDA produced, the greater was the depletion of tissue antioxidants secondary to oxygen free radical formation during reoxygenation. RESULTS: There was no difference in the prebypass antioxidant reserve capacity between cyanotic and acyanotic hearts (492 +/- 72 versus 439 +/- 44 nmol MDA/g protein). However, after the initiation of CPB without leukocyte filtration, MDA production rose markedly in the cyanotic (groups 1 and 2) as compared with the acyanotic hearts (322% versus 40%; p < 0.05), indicating a depletion of antioxidants. In cyanotic hearts, initiating CPB at an FiO2 of 1.0 (group 1) resulted in increased MDA production (407% versus 227%) as compared with hearts in which CPB was initiated at an FiO2 of 0.21 (group 2), indicating a greater generation of oxygen free radicals in group 1. Conversely, there was only a minimal increase in MDA production in 8 of the 21 infants (group 3) in whom white blood cells were effectively filtered (19% versus 322%; p < 0.05). CONCLUSIONS: First, increased amounts of oxygen free radicals are generated in cyanotic infants with the initiation of CPB. Second, this production is reduced by initiating CPB at an FiO2 of 0.21 or by effectively filtering white blood cells. Third, these changes parallel those seen in the acute experimental model, validating its use for future study.

Myocardial protection in normal and hypoxically stressed neonatal hearts: the superiority of blood versus crystalloid cardioplegia.

OBJECTIVES: Blood cardioplegia predominates in the adult because it provides superior myocardial protection, especially in the ischemically stressed heart. However, the superiority of blood over crystalloid cardioplegia in the pediatric population is unproved. Furthermore, because many pediatric hearts undergo a preoperative stress such as hypoxia, it is important to compare the different methods of protection in both normal and hypoxic hearts. METHODS: Twenty neonatal piglets were supported by cardiopulmonary bypass and subjected to 70 minutes of cardioplegic arrest. Of 10 nonhypoxic hearts, five (group 1) were protected with blood cardioplegia and five (group 2) with crystalloid cardioplegia (St. Thomas' Hospital solution). Ten other piglets underwent 60 minutes of ventilator hypoxia (inspired oxygen concentration 8% to 10%) before cardioplegic arrest. Five (group 3) were then protected with blood cardioplegia and the other five (group 4) with crystalloid cardioplegia. Myocardial function was assessed by means of pressure volume loops and expressed as a percentage of control. Coronary vascular resistance was measured with each infusion of cardioplegic solution. RESULTS: No difference was noted between blood (group 1) or crystalloid cardioplegia (group 2) in nonhypoxic hearts regarding systolic function (end-systolic elastance 104% vs 103%), diastolic stiffness (156% vs 159%), preload recruitable stroke work (102% vs 101%), or myocardial tissue edema (78.9% vs 78.9%). Conversely, in hearts subjected to a hypoxic stress, blood cardioplegia (group 3) provided better protection than crystalloid cardioplegia (group 4) by preserving systolic function (end-systolic elastance 106% vs 40%; p < 0.05) and preload recruitable stroke work (103% vs 40%; p < 0.05); reducing diastolic stiffness (153% vs 240%; p < 0.05) and myocardial tissue edema (79.6% vs 80.1%); and preserving vascular function, as evidenced by unaltered coronary vascular resistance (p < 0.05). CONCLUSION: This study demonstrates that (1) blood or crystalloid cardioplegia is cardioprotective in hearts not compromised by preoperative hypoxia and (2) blood cardioplegia is superior to crystalloid cardioplegia in hearts subjected to the preoperative stress of acute hypoxia.

Prevention of the hypoxic reoxygenation injury with the use of a leukocyte-depleting filter.

OBJECTIVES: Recent studies have shown that an injury occurs when the hypoxic heart is suddenly reoxygenated (as occurs with cardiopulmonary bypass), resulting in myocardial depression, impaired oxygenation, and increased pulmonary vascular resistance. We hypothesize that this injury is, in part, due to oxygen-derived radicals produced by activated white cells and may therefore be ameliorated by limiting leukocytes in the bypass circuit. METHODS: Fifteen neonatal piglets underwent 60 minutes of ventilator hypoxia (inspired oxygen fraction 8% to 10%), followed by reoxygenation with cardiopulmonary bypass at an inspired oxygen fraction of 100% for 90 minutes. In nine piglets (group 1) our routine bypass circuit was used with no modifications, and in six piglets (group 2) a leukocyte-depleting filter (Pall BC-1; Pall Biomedical Products Corporation, Glencoe, N.Y.) was inserted in the arterial line to lower the neutrophil count. Six additional piglets underwent 90 minutes of bypass without hypoxia (cardiopulmonary bypass controls). Postbypass myocardial and pulmonary function was assessed by pressure volume loops, arterial/alveolar ratio, and pulmonary vascular resistance index. Results are expressed as a percentage of control. RESULTS: By comparison with group 1 piglets (reoxygenation without a filter), hypoxic piglets undergoing reoxygenation with a leukocyte-depleting filter (group 2) had improved myocardial systolic function (88% vs 52%; p < 0.05), diastolic compliance (175% vs 275%; p < 0.05), and preload recruitable stroke work (91% vs 54%; p < 0.05); had better preservation of the arterial/alveolar ratio (97% vs 74%; p < 0.05); and had less increase in pulmonary vascular resistance (229% vs 391%; p < 0.05). Furthermore, leukocyte filtration prevented adenosine triphosphate depletion or a change in tissue antioxidants. Conversely, unprotected piglets (group 1) exhibited lower levels of adenosine triphosphate and significant loss of tissue antioxidants. Indeed, the results in the leukocyte-filtered piglets (group 2) were nearly identical to those of piglets subjected to bypass without hypoxia (controls). CONCLUSIONS: (1) This study demonstrates that a major component of the injury that occurs when the hypoxic heart is abruptly reoxygenated is caused by oxygen radicals produced by white blood cells; (2) this injury can be prevented by a leukocyte-depleting filter; and (3) avoidance of this injury improves postbypass myocardial and pulmonary function. These data suggest that leukocyte depletion should be used routinely in all children undergoing operations for cyanotic heart disease or extracorporeal membrane oxygenation.

Crystallization and preliminary X-ray analysis of the thermostable alkaline-tolerant xylanase from Bacillus stearothermophilus T-6.

The extracellular thermostable xylanase (XT-6) produced by the thermophilic bacterium Bacillus stearothermophilus T-6 was shown to bleach pulp optimally at pH 9 and 338 K, and was successfully used in a large-scale biobleaching mill trial. The xylanase gene was cloned and sequenced. The mature enzyme consists of 379 amino acids with a calculated molecular weight of 43,808 and pI of 9.0. Crystallographic studies of XT-6 were initiated to study the mechanism of catalysis as well as to provide a structural basis for rational introduction of enhanced thermostability by site-specific mutagenesis. This report describes the crystallization and preliminary crystallographic characterization of the native XT-6 enzyme. The most suitable crystals were obtained by the vapor-diffusion method using ammonium sulfate and 2-methyl-2,4-pentanediol as an organic additive. The crystals belong to a primitive trigonal crystal system (space group P3(1) or P3(2)) with room-temperature cell dimensions of a = b = 114.9 and c = 122.6 A. At 103 K the volume of the unit cell decreased significantly with observed dimensions of a = b = 112.2 and c = 122.9 A. These crystals are mechanically strong and diffract X-rays to better than 2.2 A resolution. The crystals exhibit considerable radiation damage at room temperature even at relatively short exposures to X-rays. A full 2.3 A resolution diffraction data set (99.8% completeness) has recently been collected on flash-frozen crystals at 103 K using synchrotron radiation. Two derivatives of XT-6 were recently prepared. In the first derivative, a unique Cys residue replaced Glu265, the putative nucleophile in the active site. The second derivative was selenomethionyl xylanase which was produced biosynthetically. These derivatives have been crystallized and the resulting crystals were shown to be isomorphous to the native crystals and diffract X-rays to comparable resolutions.

The relationship between calcium and magnesium in pediatric myocardial protection.

OBJECTIVE: We previously demonstrated that calcium can be harmful to the hypoxic neonatal heart. Despite the fact that magnesium inhibits membrane transport of calcium, few studies have examined whether magnesium can prevent the deleterious effects of calcium in cardioplegic solutions. METHODS: Twenty neonatal piglets (5 to 18 days old) underwent 60 minutes of ventilator hypoxia (inspired oxygen fraction 8% to 10%) followed by reoxygenation with the use of cardiopulmonary bypass before cardioplegic arrest to produce a clinically relevant hypoxic "stress" injury. The aorta was then crossclamped for 70 minutes with multidose blood cardioplegia. Ten piglets received a hypocalcemic (0.2 to 0.4 mmol/L) cardioplegic solution without (group 1, n = 5) or with magnesium (10 mEq/L) (group II, n = 5) supplementation. Ten other piglets were protected with a normocalcemic (1.0 to 1.2 mmol/L) cardioplegic solution without (group III, n = 5) or with magnesium (group IV, n = 5). Myocardial function was assessed by means of pressure volume loops and expressed as a percentage of control. Coronary vascular resistance was assessed during each cardioplegic infusion. RESULTS: Adding magnesium to a hypocalcemic cardioplegic solution (groups I and II) had no effect: Both groups had complete preservation of postbypass systolic function (end-systolic elastance 101% vs 104%) and preload recruitable stroke work (101% vs 102%), minimal increase in diastolic stiffness (159% vs 153%), and no difference in myocardial tissue edema (78.8% vs 78.9%) or coronary vascular resistance. Conversely, when a normocalcemic cardioplegic solution was administered without magnesium supplementation (group III), the results were markedly poorer than results obtained with magnesium supplementation (group IV). Without magnesium, there was a marked reduction in postbypass systolic function (end-systolic elastance 49% vs 101%; p < 0.05), increased diastolic stiffness (276% vs 162%; p < 0.05), decreased preload recruitable stroke work (53% vs 102%; p < 0.05), increased myocardial tissue edema (80.0% vs 78.9%; p < 0.05), and a rise in coronary vascular resistance (p < 0.05). Magnesium supplementation of the normocalcemic cardioplegic solution, by contrast, resulted in complete functional recovery. CONCLUSIONS: This study demonstrates that (1) magnesium does not alter the cardioprotective effects of a hypocalcemic cardioplegic solution, (2) a normocalcemic cardioplegic solution is detrimental to neonatal myocardium subjected to a previous hypoxic stress, and (3) magnesium supplementation of normocalcemic cardioplegic solutions prevents the deleterious effects of calcium.

Myocardial protection in normal and hypoxically stressed neonatal hearts: the superiority of hypocalcemic versus normocalcemic blood cardioplegia.

OBJECTIVES: The ideal cardioplegic calcium (Ca+2) concentration in newborns continues to be debated. Most studies examining cardioplegia calcium concentrations have been done with a nonclinical model (i.e., isolated heart preparation), the results of which may not be clinically applicable, and they have not examined the effect of calcium concentration in a clinically relevant stressed (hypoxic) heart. METHODS: Twenty neonatal piglets 5 to 18 days old were placed on cardiopulmonary bypass, and their aortas were crossclamped for 70 minutes with hypocalcemic or normocalcemic multidose blood cardioplegic infusions. Group 1 (n = 5; low Ca+2, 0.2 to 0.4 mmol/L) and group 2 (n = 5; normal Ca+2, 1.0 to 1.3 mmol/L) were nonhypoxic (uninjured) hearts. Ten other piglets were first ventilated at an FiO2 of 8% to 10% (O2 saturation 65% to 70%) for 60 minutes (i.e., causing hypoxia) and then reoxygenated at an FiO2 of 100% with cardiopulmonary bypass, which produces a clinically relevant stress injury. They then underwent cardioplegic arrest (as described above) with a hypocalcemic (n = 5, group 3) or normocalcemic (n = 5, group 4) blood cardioplegic solution. Myocardial function was assessed with pressure volume loops and expressed as a percentage of control values. Coronary vascular resistance was measured during each cardioplegic infusion. All values were reported as the mean +/- standard error. RESULTS: In nonhypoxic hearts (groups 1 and 2), good myocardial protection was achieved at either concentration of cardioplegia calcium, as demonstrated by preservation of postbypass systolic function (104% vs 99% end-systolic elastance), minimally increased diastolic stiffness (152% vs 162%), no difference in myocardial water (78.9% vs 78.9%), and no change in adenosine triphosphate levels or coronary vascular resistance. Low-calcium blood cardioplegia solution repaired the hypoxic reoxygenation injury in stressed hearts (group 3), resulting in no statistical difference in myocardial function, coronary vascular resistance, or adenosine triphosphate levels compared with nonhypoxic hearts (groups 1 and 2). Conversely, when a normocalcemic cardioplegia solution was used in hypoxic hearts (group 4), there was marked reduction in postbypass systolic function (49% +/- 4% end-systolic elastance; p < 0.05), increased diastolic stiffness (276% +/- 9%; p < 0.05), increased myocardial water (80.1% +/- 0.2%; p < 0.05), rise in coronary vascular resistance (p < 0.05), and lower adenosine triphosphate levels compared with groups 1, 2, and 3. CONCLUSIONS: This study demonstrates that, in the clinically relevant, intact animal model, good myocardial protection is independent of cardioplegia calcium concentration in nonhypoxic (noninjured) hearts; hypoxic (stressed) hearts are extremely sensitive to the cardioplegic calcium concentration; and normocalcemic cardioplegia is detrimental to neonatal myocardium subjected to a preoperative hypoxic stress.

The use of methotrexate in polymyalgia rheumatica.

OBJECTIVE: To examine the efficacy and steroid sparing potential of methotrexate (MTX) in the treatment of polymyalgia rheumatica (PMR) without giant cell arteritis. METHODS: 43 patients with PMR were treated with MTX 7.5 mg/wk for 3 months. This dose was raised to 10 and finally 12.5 mg/wk MTX if the desired response was not obtained. RESULTS: No patient benefitted clinically or statistically from treatment with MTX. CONCLUSION: MTX is of no benefit in the treatment of PMR.

Zinc-directed inhibitors for zinc proteinases.

Zinc proteinases have been recognized as a distinct class of proteolytic enzymes in which at least one ion of zinc is involved directly in catalysis. This family includes a growing number of biologically important enzymes which are attractive targets for rational drug design. In this paper we examine the special features of the zinc binding environment of these enzymes in order to gain information which could be useful in the preparation of 'zinc-directed' selective inhibitors. Carboxypeptidase A (CPA) is presented as a model for one class of zinc proteinases, and the active-site zinc and its interactions are examined with the primary focus on geometrical considerations. The three-dimensional structure of the native and apoenzyme are discussed, together with the high-resolution structure of several enzyme-inhibitor complexes. This paper will first present a structural analysis of CPA derivatives and then discuss a series of zinc model compounds which have been prepared and characterized in order to examine the ligand and geometrical preferences of the zinc in an unstrained system. X-ray crystallography (macromolecular and small molecule) is the main experimental method used for the structural analyses, while complementary computational methods have been used for the examination of electrostatic potentials. The results from the various experimental efforts are assembled in order to draw general conclusions on the potential use of the zinc ion as the primary target for inhibitor binding. The results of these studies suggest that the zinc ion is important for both the binding and the catalytic activation of the substrate as well as for stabilization of the tetrahedral reaction intermediate.

Myocardial cytosolic calcium accumulation during ischemia/reperfusion: the effects of aging and cardioplegia.

Cytosolic calcium in the myocardium is rapidly accumulated during ischemia and has been correlated with the attenuation of functional recovery in the myocardium. The aged myocardium is more sensitive to ischemia and accumulates significantly more cytosolic calcium than either the newborn or the mature myocardium. Modification of the age-related propensity to increased cytosolic calcium accumulation may be achieved through the use of magnesium or potassium/magnesium cardioplegia. Improved postischemic ventricular function obtained with magnesium or potassium/magnesium cardioplegia may have important implications in the reduction of myocardial morbidity and mortality.

Fura 2 determination of [Ca2+]i in isolated perfused heart using R wave-gated electromechanical shutters.

We describe a novel and relatively inexpensive spectrofluorescence system that supplies rapidly alternating wavelengths to either a standard cuvette or an isolated perfused heart. Its use is illustrated by determining changes in cytosolic intracellular Ca2+ concentration ([Ca2+]i) by using the Ca(2+)-sensitive fluorescent dye fura 2 in a rabbit heart preparation. The system uses two precision electromechanical shutters (capable of gating with respect to the electrocardiographic R wave for signal averaging) allowing alternate fura 2 excitation wavelengths (340 and 380 nm) without moving optical components and uses a fiber optic for conducting excitation and collecting epifluorescence. Sample recordings tracing the [Ca2+]i transient in an entire cardiac cycle and in capturing specific isolated regions (diastole and systole) of the cycle are presented. Limitations of this low-cost but easily implemented system are discussed.