Pitfalls in Echocardiography: Coarctation of the Aorta Presenting as Dilated Cardiomyopathy (DCM).

Purpose: The morphologic spectrum of aortic coarctation extends from discrete isthmic obstruction to tubular hypoplasia of the entire aortic arch. Neonates with coarctation frequently present with congestive heart failure and critically reduced perfusion of the descending aorta following ductal closure. During the recent years we observed several infants with coarctation who presented beyond the neonatal period with dilated cardiomyopathy (DCM). We reviewed our patients with coarctation to determine whether this presentation represents an exception or is relevant for the differential diagnosis of children with DCM. Materials and Methods: From 1/2001 to 12/2013 74 babies with isolated coarctation were diagnosed in our institution. 50 patients presented in the neonatal period and 24 patients beyond the first month. Results: 5/74 infants presented after the neonatal period with poorly contractile, dilated left ventricles. Echocardiographic detection of the coarctation was facilitated by application of the ductal view and by Doppler interrogation of the celiac artery revealing a significantly diminished systolic flow velocity. All patients underwent resection of the coarctation and end-to-end anastomosis of the aorta. Postoperative normalization of left ventricular function was observed within a median interval of 2 months. Conclusion: Coarctation of the aorta presenting as DCM accounted for 21 % of our infants with coarctation who presented beyond the neonatal period and 7 % of those in the first year of life. The stenosis was difficult to detect because of its distal location and normal configuration of the aortic arch. Isthmic coarctation should be included in the differential diagnosis of infants with DCM and requires careful examination of the isthmic region in these children.

Severe chest pain caused by Mycoplasma myocarditis in an adolescent patient.

Intermittent chest pain is a common symptom in adolescent patients, which can be attributed to musculoskeletal disorders in the majority of cases. While most patients have a benign course of their pain episodes, the differential diagnosis includes diseases which are associated with serious and even life-threatening complications. Therefore, careful examination and systematic diagnostic evaluation is important. We report on an adolescent, who presented with severe chest pain and distinct electrocardiographic findings caused by Mycoplasma pneumoniae myocarditis.

Factor X deficiency and intracranial bleeding: who is at risk?

Very few mutations of the gene encoding for coagulation factor X (FX) have been found associated with intracranial haemorrhage (ICH) due to FX deficiency (FXD). No guidelines exist as to when prophylaxis in FXD should be started and how patients at risk for ICH can be identified. We report on a novel mutation causative for ICH in a family of Iranian origin and provide a summary of all published mutations in the FX gene related to ICH. The index patient is an infant with umbilical bleeding requiring blood transfusion in the postnatal period. The international normalized ratio (6.01) and activated partial thromboplastin time (117 s) were prolonged. Coagulation factor analysis was normal except for FX activity (<1%). At 4 months, the child suffered a spontaneous severe intracranial haemorrhage. The child was the product of a consanguineous union. Four of five available family members from three generations displayed minor bleeding symptoms and mildly reduced FX. Sequencing of FX gene demonstrated homozygosity for a novel duplication A (c.1402_1403dupA)* in exon 8 and heterozygosity in four family members. We compare this case to all 15 patients with FXD and ICH and their 11 known mutations described so far. This case illustrates a pattern of FXD (a male neonate with umbilical or gastrointestinal bleeding, very low FX:C (<1%) and an underlying homozygous genotype) who may be at high risk for ICH. In these cases, we recommend to start early prophylactic substitution of FX to prevent a possible life-threatening haemorrhage.

Increased apoptosis of Huntington disease lymphoblasts associated with repeat length-dependent mitochondrial depolarization.

Huntington disease (HD) is a genetically dominant condition caused by expanded CAG repeats coding for glutamine in the HD gene product huntingtin. Although HD symptoms reflect preferential neuronal death in specific brain regions, huntingtin is expressed in almost all tissues, so abnormalities outside the brain might be expected. Although involvement of nuclei and mitochondria in HD pathophysiology has been suggested, specific intracellular defects that might elicit cell death have been unclear. Mitochondria dysfunction is reported in HD brains; mitochondria are organelles that regulates apoptotic cell death. We now report that lymphoblasts derived from HD patients showed increased stress-induced apoptotic cell death associated with caspase-3 activation. When subjected to stress, HD lymphoblasts also manifested a considerable increase in mitochondrial depolarization correlated with increased glutamine repeats.

Interictal and postictal performances on dichotic listening test in children with focal epilepsy.

Dichotic listening test (DL) is an important tool to disclose speech dominance in healthy subjects and in clinical cases. The aim of this study was to probe if focal epilepsy in children reveals a corresponding suppression of the ear reports contralateral to seizure onset site. Thus, 15 children and adolescents with clinically and electroencephalographically diagnosed focal epilepsy selected for left-hemisphere speech dominance without mental retardation were compared to matched controls according to age, gender, IQ and handedness. All children were assessed with DL for three times: Interictally (t(0)), postictally 5' (t(1)) and 1h (t(2)). At t(0), all groups revealed a right ear advantage (REA), indicating a left-hemisphere speech dominance. There was a continuous increase in right correct score (REC) over the trials for normal controls. Five minutes postictally, there was an abrupt decrease in REC with a sustained left ear correct score (LEC) for children with epilepsy, independent of which side suffered from seizures. This effect was maintained even after 1h. Thus, in children with left-hemisphere speech dominance the epileptic discharges caused a suppression of REC regardless of origin. The seizures may have a prolonged impact on attention and auditory perception for a considerable time after consciousness has been regained.

Neonatal Marfan syndrome: unusually large deletion of exons 24-26 of FBN1 associated with poor prognosis.

Neonatal Marfan syndrome is a very rare subset of the classical Marfan syndrome with pronounced phenotypic expression especially of the cardiovascular manifestations. It is associated with a very poor prognosis, with approximately 50% of affected infants dying from cardiac failure during the first year of life. We present a newborn with the classical phenotype of neonatal Marfan syndrome. Within few hours after birth, progressive and refractory heart failure developed. Postmortal molecular study revealed an unusually large deletion of exons 24-26 within the so-called neonatal region of the gene FBN1, which might explain the unfavourable course of the disease in our patient.

The Wada test in Austrian, Dutch, German, and Swiss epilepsy centers from 2000 to 2005: a review of 1421 procedures.

Twenty-six Austrian, Dutch, German, and Swiss epilepsy centers were asked to report on use of the Wada test (intracarotid amobarbital procedure, IAP) from 2000 to 2005 and to give their opinion regarding its role in the presurgical diagnosis of epilepsy. Sixteen of the 23 centers providing information had performed 1421 Wada tests, predominantly the classic bilateral procedure (73%). A slight nonsignificant decrease over time in Wada test frequency, despite slightly increasing numbers of resective procedures, could be observed. Complication rates were relatively low (1.09%; 0.36% with permanent deficit). Test protocols were similar even though no universal standard protocol exists. Clinicians rated the Wada test as having good reliability and validity for language determination, whereas they questioned its reliability and validity for memory lateralization. Several noninvasive functional imaging techniques are already in use. However, clinicians currently do not want to rely solely on noninvasive functional imaging in all patients.

Acquired von Willebrand syndrome in congenital heart disease surgery: results from an observational case-series.

Essentials Bleeding complications during congenital heart disease surgery in neonatal age are very common. We report the perioperative incidence of acquired von Willebrand syndrome (aVWS) in 12 infants. aVWS was detected in 8 out of 12 neonates and infants intraoperatively after cardiopulmonary bypass. Ten patients received von Willebrand factor concentrate intraoperatively and tolerated it well. SUMMARY: Background Cardiac surgery of the newborn and infant with complex congenital heart disease (CHD) is associated with a high rate of intraoperative bleeding complications. CHD-related anatomic features such as valve stenoses or patent arterial ducts can lead to enhanced shear stress in the blood stream and thus cause acquired von Willebrand syndrome (aVWS). Objective To evaluate the intraoperative incidence and impact of aVWS after cardiopulmonary bypass (CPB) in neonates and infants with complex CHD. Patients/Methods We conducted a survey of patients aged < 12 months undergoing complex cardiac surgery in our tertiary referral center. Twelve patients, whose blood samples were analyzed for aVWS before CPB and immediately after discontinuation of CPB on a routine basis, were eligible for the analysis. von Willebrand factor antigen (VWF:Ag), ristocetin cofactor activity (VWF:RCo), collagen binding activity (VWF:CB), VWF:multimers and factor VIII activity (FVIII:C) were determined. Results aVWS was diagnosed by VWF multimer analysis in 10 out of 12 patients (83%) prior to surgery and intraoperatively at the end of CPB in 8 out of 12 patients (66%). Ten patients received VWF/FVIII concentrate intraoperatively as individual treatment attempts during uncontrolled bleeding. They tolerated it well without intraoperative thrombotic events. One patient suffered a transient postoperative cerebral sinuous vein thrombosis. Conclusions aVWS is of underestimated incidence in complex CHD surgery. These data may offer a new approach to reduce the risk of severe bleedings and to achieve hemostasis during high-risk pediatric cardiac surgery by tailoring the substitution with von Willebrand factor concentrate.

[A life-threatening cardiomyopathy following port-a-cath infection under immune tolerance therapy]. / Lebensbedrohliche Kardiomyopathie als Folge einer Portkatheterinfektion bei Immuntoleranztherapie.

The development of inhibitors in patients with severe haemophilia A is a serious complication requiring long term immune tolerance therapy (ITT). ITT frequently requires implantable central venous access, mostly port catheters. Their use may be complicated by thrombosis and infection. We report on an 18 year old patient with severe haemophilia A who had developed a high-titre factor VIII inhibitor in the age of five years. ITT required the implantation of a port system. The postoperative course was complicated by severe septicaemia with congestive cardiac failure. The port catheter was removed due to recurrent fever after 26 days. Our patient developed dilative cardiomyopathy. ITT had to be stopped and was replaced by on demand therapy with an activated prothrombin complex concentrate. Cardiomyopathy resulted in congestive heart failure, severe ventricular arrhythmias and the death of the young man. In patients with haemophilia, dilative cardiomyopathy and development of inhibitors the possibility of cardiac transplantation should be evaluated before increasing inhibitors and the development of pulmonary hypertension exclude this therapeutical option.

Activation of Bacillus licheniformis alpha-amylase through a disorder-->order transition of the substrate-binding site mediated by a calcium-sodium-calcium metal triad.

BACKGROUND: The structural basis as to how metals regulate the functional state of a protein by altering or stabilizing its conformation has been characterized in relatively few cases because the metal-free form of the protein is often partially disordered and unsuitable for crystallographic analysis. This is not the case, however, for Bacillus licheniformis alpha-amylase (BLA) for which the structure of the metal-free form is available. BLA is a hyperthermostable enzyme which is widely used in biotechnology, for example in the breakdown of starch or as a component of detergents. The determination of the structure of BLA in the metal-containing form, together with comparisons to the apo enzyme, will help us to understand the way in which metal ions can regulate enzyme activity. RESULTS: We report here the crystal structure of native, metal-containing BLA. The structure shows that the calcium-binding site which is conserved in all alpha-amylases forms part of an unprecedented linear triadic metal array, with two calcium ions flanking a central sodium ion. A region around the metal triad comprising 21 residues exhibits a conformational change involving a helix unwinding and a disorder-->order transition compared to the structure of metal-free BLA. Another calcium ion, not previously observed in alpha-amylases, is located at the interface between domains A and C. CONCLUSIONS: We present a structural description of a major conformational rearrangement mediated by metal ions. The metal induced disorder-->order transition observed in BLA leads to the formation of the extended substrate-binding site and explains on a structural level the calcium dependency of alpha-amylases. Sequence comparisons indicate that the unique Ca-Na-Ca metal triad and the additional calcium ion located between domains A and C might be found exclusively in bacterial alpha-amylases which show increased thermostability. The information presented here may help in the rational design of mutants with enhanced performance in biotechnological applications.

A novel strategy for inhibition of alpha-amylases: yellow meal worm alpha-amylase in complex with the Ragi bifunctional inhibitor at 2.5 A resolution.

BACKGROUND: alpha-Amylases catalyze the hydrolysis of alpha-D-(1,4)-glucan linkages in starch and related compounds. There is a wide range of industrial and medical applications for these enzymes and their inhibitors. The Ragi bifunctional alpha-amylase/trypsin inhibitor (RBI) is the prototype of the cereal inhibitor superfamily and is the only member of this family that inhibits both trypsin and alpha-amylases. The mode of inhibition of alpha-amylases by these cereal inhibitors has so far been unknown. RESULTS: The crystal structure of yellow meal worm alpha-amylase (TMA) in complex with RBI was determined at 2.5 A resolution. RBI almost completely fills the substrate-binding site of TMA. Specifically, the free N terminus and the first residue (Ser1) of RBI interact with all three acidic residues of the active site of TMA (Asp185, Glu222 and Asp287). The complex is further stabilized by extensive interactions between the enzyme and inhibitor. Although there is no significant structural reorientation in TMA upon inhibitor binding, the N-terminal segment of RBI, which is highly flexible in the free inhibitor, adopts a 3(10)-helical conformation in the complex. RBI's trypsin-binding loop is located opposite the alpha-amylase-binding site, allowing simultaneous binding of alpha-amylase and trypsin. CONCLUSIONS: The binding of RBI to TMA constitutes a new inhibition mechanism for alpha-amylases and should be general for all alpha-amylase inhibitors of the cereal inhibitor superfamily. Because RBI inhibits two important digestive enzymes of animals, it constitutes an efficient plant defense protein and may be used to protect crop plants from predatory insects.

Antitumor activity of murine neutrophils demonstrated by cytometric analysis.

The cytostatic and cytolytic activities of activated polymorphonuclear neutrophils (PMNs) against YAC-1 lymphoma target cells were examined using multiparameter flow cytometric analysis. PMNs were resolved from tumor cells by 90 degrees light scatter. The number of surviving tumor cells was determined by adding a known concentration of fluorescent latex particles to the fixed cell suspension immediately prior to analysis and counting the particles simultaneously with the cells. Cell cycle progression of the YAC-1 target was studied by dual parameter analysis of DNA content and bromodeoxyuridine incorporation into tumor cell DNA either prior to or following addition of PMNs. The results indicate that activated PMNs effectively kill tumor cells within the first 24 h of coculture. However, between 24 and 48 h, tumor cells which escape destruction resume growth and eventually reach a growth rate greater than control cells.

Crystal structure of calcium-depleted Bacillus licheniformis alpha-amylase at 2.2 A resolution.

The three-dimensional structure of the calcium-free form of Bacillus licheniformis alpha-amylase (BLA) has been determined by multiple isomorphous replacement in a crystal of space group P4(3)2(1)2 (a = b = 119.6 A, c = 85.4 A). The structure was refined using restrained crystallographic refinement to an R-factor of 0.177 for 28,147 independent reflections with intensities FObs > 0 at 2.2 A resolution, with root mean square deviations of 0.008 A and 1.4 degrees from ideal bond lengths and bond angles, respectively. The final model contains 469 residue, 237 water molecules, and one chloride ion. The segment between Trp182 and Asn192 could not be located in the electron density, nor could the N and C termini. Cleavage of the calcium-free form of BLA was observed after Glu189, due to a Glu-C endopeptidase present in trace amounts in the preparation. BLA did not crystallize without this cleavage under the conditions applied. BLA exhibits the characteristic overall topological fold observed for other alpha-amylases and related amylolytic enzymes: a central domain A containing an alpha/beta-barrel with a large protrusion between beta-strand 3 and alpha-helix 3 (domain B) and a C-terminal greek key motif (domain C). Unlike in the other enzymes, domain B possesses a beta-sheet made up of six loosely connected, twisted beta-strands forming a kind of a barrel with a large hole in the interior. Topological comparisons to TAKA-amylase, pig pancreatic alpha-amylase and cyclodextrin glycosyltransferase reveal a very high structural equivalence for large portions of the proteins and an exceptionally pronounced structural similarity for calcium binding, chloride binding and the active site. None of the theories proposed to explain the enhanced thermostability of BLA showed a satisfactory correlation with the three-dimensional structure. Instead, sequence comparisons to the less thermostable bacterial alpha-amylase from Bacillus amyloliquefaciens (BAA) indicate that some ionic interactions present in BLA, but which cannot be formed in BAA, might be responsible for the enhanced thermostability of BLA.

The crystal structure of porcine pancreatic alpha-amylase in complex with the microbial inhibitor Tendamistat.

The crystal structure of the complex formed between the 498 amino acid residue porcine pancreatic alpha-amylase (PPA) and the 74 amino acid residue inhibitor Tendamistat secreted from Streptomyces tendae, has been determined by multiple isomorphous replacement in a crystal of space group P6(5)22 (a = b = 77.7 A, c = 359.5 A). The model has been refined to an R-factor of 0.194 by Powell minimization applying strong energy constraints based on 17,964 independent reflections in the 7 to 2.5 A resolution range, and obeys standard geometry within 0.011 A in bond lengths and 1.78 degrees in bond angles. The final model consists of all 496 amino acid residues of PPA, 71 amino acid residues of Tendamistat (without the three N-terminal residues), one calcium ion, one chloride ion and 167 water molecules. PPA exhibits the same topological fold in the complex as the uncomplexed PPA recently published by others. About 30% of the water-accessible surface of Tendamistat is in contact with PPA. Four segments of the polypeptide chain, with a total of 15 amino acid residues, are involved in the binding. One segment containing the staggered side-chains of the triplet Trp18, Arg19, Tyr20, typical for this class of inhibitors, binds into the catalytic site. The other segments fill out the groove in the PPA molecule, which also binds the carbohydrate inhibitor acarbose and is assumed to be the substrate-binding region. This extended interaction between Tendamistat and alpha-amylase explains the very high inhibition constant of about 9 x 10(-12) M.

Crystal structure analysis and molecular model of a complex of citrate synthase with oxaloacetate and S-acetonyl-coenzyme A.

The crystal structure of the complex of pig heart citrate synthase and oxaloacetate in the presence of the potent inhibitor S-acetonyl coenzyme A has been determined at a nominal resolution of 2.9 A by Patterson search techniques and refined by restrained crystallographic refinement. The complex crystallizes in the presence of polyvinylpyrrolidone in space group P4(3)2(1)2 with a = 101.5 A and c = 224.6 A, with one dimeric molecule of molecular weight 100,000 in the asymmetric unit. The crystallographic R factor is 0.194 for the 14,332 unique reflections between 6.0 and 2.9 A resolution. The structures of two forms of citrate synthase in the presence and absence of product molecules have been determined recently and shown to differ in the relative arrangement of the large and small domains ("closed" and "open" forms). The third crystal form described here is also closed, but there is substantial rearrangement within the small domain relative to either of the other crystal forms. We conclude that this is a third structural state of the enzyme, and catalytic activity of the enzyme depends on structural changes during the course of the reaction affecting domain conformation also. The three structures are compared, and it is shown that the large domain is considerably more rigid than the small domain. The conformation of the small domain adapts to the ligand. The inhibitor, and the "coenzyme-A-binding segment" of the enzyme are disordered. No electron density is observed for the inhibitor, and only weak density for the coenzyme-A-binding segment. Electron density for oxaloacetate is well defined. It binds in a very similar manner to citrate.

Carbohydrate and protein-based inhibitors of porcine pancreatic alpha-amylase: structure analysis and comparison of their binding characteristics.

The crystal structures of porcine pancreatic alpha-amylase isozyme II (PPA II) in its free form and complexed with the trestatin A derived pseudo-octasaccharide V-1532 have been determined using Patterson search techniques at resolutions of 2.3 and 2.2 angstroms, respectively. Seven rings of the competitive inhibitor V-1532 could be detected in the active site region as well as two maltose units in secondary binding sites on the surface. V-1532 occupies the five central sugar binding subsites similar to the PPA/acarbose structure. A sixth ring exists at the reducing end, connecting two symmetry related PPA molecules. The seventh moiety, a 6-hydroxymethylconduritol ring, is located at the non-reducing end. The electron density for this ring is relatively weak, indicating considerable disorder. This study shows that PPA is able to accommodate more than five rings in the active site region, but that additional rings would increase the binding affinity only slightly, which is in accordance with kinetic experiments. A comparison of the structures of free PPA, PPA/V-1532 and PPA/Tendamistat shows the characteristic conformational changes that accompany inhibitor binding and distinguish pseudo-oligosaccharide inhibitors from proteinaceous inhibitors. Although both classes of inhibitors block the sugar binding subsites in the active site region, the extreme specificity and binding affinity of the proteinaceous inhibitors is probably due to an intricate interaction pattern involving areas further away from the catalytic center.

Crystal structure determination, refinement and the molecular model of the alpha-amylase inhibitor Hoe-467A.

The crystal and molecular structure of the alpha-amylase inhibitor Hoe-467A has been determined and refined at high resolution. The polypeptide chain is folded in two triple-stranded sheets, which form a barrel. The topology of folding is as found in the immunoglobulin domains. The amino acid triplet Trp18-Arg19-Tyr20 has an exceptional conformation and position in the molecule and is possibly involved in inhibitory activity.

Probing structural determinants specifying high thermostability in Bacillus licheniformis alpha-amylase.

Bacillus licheniformis alpha-amylase (BLA) is a starch-degrading enzyme that is highly thermostable although it is produced by a rather mesophilic organism. Over the last decade, the origin of BLA thermal properties has been extensively investigated in both academic and industrial laboratories, yet it is poorly understood. Here, we have used structure-based mutagenesis in order to probe the role of amino acid residues previously proposed as being important for BLA thermostability. Residues involved in salt-bridges, calcium binding or potential deamidation processes have been selected and replaced with various amino acids using a site-directed mutagenesis method, based on informational suppression. A total of 175 amylase variants were created and analysed in vitro. Active amylase variants were tested for thermostability by measuring residual activities after incubation at high temperature. Out of the 15 target residues, seven (Asp121, Asn126, Asp164, Asn192, Asp200, Asp204 and Ala269) were found to be particularly intolerant to any amino acid substitutions, some of which lead to very unstable mutant enzymes. By contrast, three asparagine residues (Asn172, Asn188 and Asn190) could be replaced with amino acid residues that significantly increase the thermostability compared to the wild-type enzyme. The highest stabilization event resulted from the substitution of phenylalanine in place of asparagine at position 190, leading to a sixfold increase of the enzyme's half-life at 80 degrees C (pH 5.6, 0.1 mM CaCl(2)). These results, combined with those of previous mutational analyses, show that the structural determinants contributing to the overall thermostability of BLA concentrate in domain B and at its interface with the central A domain. This region contains a triadic Ca-Na-Ca metal-binding site that appears extremely sensitive to any modification that may alter or reinforce the network of electrostatic interactions entrapping the metal ions. In particular, a loop spanning from residue 178 to 199, which undergoes pronounced conformational changes upon removal of calcium, appears to be the key feature for maintaining the enzyme structural integrity. Outside this region, most salt-bridges that were destroyed by mutations were found to be dispensable, except for an Asp121-Arg127 salt-bridge that contributes to the enhanced thermostability of BLA compared to other homologous bacterial alpha-amylases. Finally, our studies demonstrate that the natural resistance of BLA against high temperature is not optimized and can be enhanced further through various means, including the removal of possibly deamidating residues.

Amino acid sequence, crystallization and structure determination of reduced and oxidized cytochrome c6 from the green alga Scenedesmus obliquus.

Cytochrome c6from the unicellular green alga Scenedesmus obliquus was sequenced, crystallized in its reduced and oxidized state and the three-dimensional structure of the protein in both redox states was determined by X-ray crystallography. Reduced cytochrome c6crystallized as a monomer in the space group P 21212, whereas the oxidized protein crystallized as a dimer in the space group P 3121. The structures were solved by molecular replacement and refined to 1. 9 and 2.0 A, respectively. Comparison of the structures of both redox states revealed only slight differences on the protein surface, whereas a distortion along the axis between the heme iron and its coordinating Met61 residue was observed. No redox-dependent movement of internal water molecules could be detected. The high degree of similarity of the surfaces and charge distributions of both redox states, as well as the dimerization of cytochrome c6as observed in the oxidized crystal, is discussed with respect to its biological relevance and its implications for the reaction mechanisms between cytochrome c6and its redox partners. The dimer of oxidized cytochrome c6may represent a molecular structure occurring in a binary complex with cytochrome b6f. This assembly might be required for the correct orientation of cytochrome c6with respect to its redox partner cytochrome b6f, facilitating the electron transfer within the complex. If the dimerization is not redox-dependent in vivo, the almost identical surfaces of both redox states do not support a long range differentiation between reduced and oxidized cyt c6, i.e. a random collision model for the formation of an electron transfer complex must be assumed.

Crystal structure of yellow meal worm alpha-amylase at 1.64 A resolution.

The three-dimensional structure of the alpha-amylase from Tenebrio molitor larvae (TMA) has been determined by molecular replacement techniques using diffraction data of a crystal of space group P212121 (a=51.24 A; b=93.46 A; c=96.95 A). The structure has been refined to a crystallographic R-factor of 17.7% for 58,219 independent reflections in the 7.0 to 1.64 A resolution range, with root-mean-square deviations of 0.008 A for bond lengths and 1.482 degrees for bond angles. The final model comprises all 471 residues of TMA, 261 water molecules, one calcium cation and one chloride anion. The electron density confirms that the N-terminal glutamine residue has undergone a post-transitional modification resulting in a stable 5-oxo-proline residue. The X-ray structure of TMA provides the first three-dimensional model of an insect alpha-amylase. The monomeric enzyme exhibits an elongated shape approximately 75 Ax46 Ax40 A and consists of three distinct domains, in line with models for alpha-amylases from microbial, plant and mammalian origin. However, the structure of TMA reflects in the substrate and inhibitor binding region a remarkable difference from mammalian alpha-amylases: the lack of a highly flexible, glycine-rich loop, which has been proposed to be involved in a "trap-release" mechanism of substrate hydrolysis by mammalian alpha-amylases. The structural differences between alpha-amylases of various origins might explain the specificity of inhibitors directed exclusively against insect alpha-amylases.