Safety Aspects of Perinatal Ultrasound.

Ultrasound safety is of particular importance in fetal and neonatal scanning. Fetal tissues are vulnerable and often still developing, the scanning depth may be low, and potential biological effects have been insufficiently investigated. On the other hand, the clinical benefit may be considerable. The perinatal period is probably less vulnerable than the first and second trimesters of pregnancy, and ultrasound is often a safer alternative to other diagnostic imaging modalities. Here we present step-by-step procedures for obtaining clinically relevant images while maintaining ultrasound safety. We briefly discuss the current status of the field of ultrasound safety, with special attention to the safety of novel modalities, safety considerations when ultrasound is employed for research and education, and ultrasound of particularly vulnerable tissues, such as the neonatal lung. This CME is prepared by ECMUS, the safety committee of EFSUMB, with contributions from OB/GYN clinicians with a special interest in ultrasound safety.

EFSUMB Clinical Safety Statement for Diagnostic Ultrasound - (2019 revision).

This document is the updated 2019 revision of the EFSUMB Clinically Safety Statement. A Safety Statement has been published by EFSUMB annually since 1994 by the Safety Committee (ECMUS) of the federation. The text is deliberately brief and gives a concise overview of safety in the use of diagnostic ultrasound.

Hepatic insulin resistance both in prediabetic and diabetic patients determines postprandial lipoprotein metabolism: from the CORDIOPREV study.

BACKGROUND/AIMS: Previous evidences have shown the presence of a prolonged and exaggerated postprandial response in type 2 diabetes mellitus (T2DM) and its relation with an increase of cardiovascular risk. However, the response in prediabetes population has not been established. The objective was to analyze the degree of postprandial lipemia response in the CORDIOPREV clinical trial (NCT00924937) according to the diabetic status. METHODS: 1002 patients were submitted to an oral fat load test meal (OFTT) with 0.7 g fat/kg body weight [12 % saturated fatty acids (SFA), 10 % polyunsaturated fatty acids (PUFA), 43 % monounsaturated fatty acids (MUFA), 10 % protein and 25 % carbohydrates]. Serial blood test analyzing lipid fractions were drawn at 0, 1, 2, 3 and 4 h during postprandial state. Postprandial triglycerides (TG) concentration at any point >2.5 mmol/L (220 mg/dL) has been established as undesirable response. We explored the dynamic response in 57 non-diabetic, 364 prediabetic and 581 type 2 diabetic patients. Additionally, the postprandial response was evaluated according to basal insulin resistance subgroups in patients non-diabetic and diabetic without pharmacological treatment (N = 642). RESULTS: Prevalence of undesirable postprandial TG was 35 % in non-diabetic, 48 % in prediabetic and 59 % in diabetic subgroup, respectively (p < 0.001). Interestingly, prediabetic patients displayed higher plasma TG and large triacylglycerol-rich lipoproteins (TRLs-TG) postprandial response compared with those non-diabetic patients (p < 0.001 and p = 0.003 respectively). Moreover, the area under the curve (AUC) of TG and AUC of TRLs-TG was greater in the prediabetic group compared with non-diabetic patients (p < 0.001 and p < 0.005 respectively). Patients with liver insulin resistance (liver-IR) showed higher postprandial response of TG compared with those patients with muscle-IR or without any insulin-resistance respectively (p < 0.001). CONCLUSIONS: Our findings demonstrate that prediabetic patients show a lower phenotypic flexibility after external aggression, such as OFTT compared with nondiabetic patients. The postprandial response increases progressively according to non-diabetic, prediabetic and type 2 diabetic state and it is higher in patients with liver insulin-resistance. To identify this subgroup of patients is important to treat more intensively in order to avoid future cardiometabolic complications.

COVID-19 pandemic on coronary artery and cerebrovascular diseases in Southern Spain: interrupted time series analysis.

OBJECTIVE: Healthcare systems have been put under intense pressure by the COVID-19 pandemic, although some studies have shown a decline in hospital admissions for cardiovascular and cerebrovascular diseases during the first and second wave of the pandemic. In addition, studies analyzing gender and procedural differences are scarce. The present study aimed to determine the impact of the pandemic on hospital admissions for acute myocardial infarction (AMI) and cerebrovascular disease (CVD) in Andalusia (Spain) and analyzed differences by gender and by percutaneous coronary interventions performed. PATIENTS AND METHODS: An interrupted time series analysis of AMI and CVD hospital admissions in Andalusia (Spain) was carried out to measure the impact of the COVID-19 outbreak. AMI and CVD cases admitted daily in public hospitals of Andalusia between January 2018 and December 2020 were included. RESULTS: During the pandemic, significant reductions in AMI [-19%; 95% confidence interval (CI): (-29%, -9%), p<0.001] and CVD [-17%; 95% CI: (-26%, -9%); p<0.01] in daily hospital admissions were observed. Differences were also produced according to the diagnosis (ST-Elevation Myocardial Infarction, Non-ST-Elevation Myocardial Infarction, other AMI and stroke), with a greater reduction in females for AMI and in males for CVD. Although there were more percutaneous coronary interventions during the pandemic, no significant reductions were observed. CONCLUSIONS: A decline in AMI and CVD daily hospital admissions during the first and second wave of COVID-19 pandemic was noted. Gender differences were observed, but no clear impact was observed in percutaneous interventions.

Prediction of phosphenes occurrence in patients undergoing photon irradiation to ocular structures due to brain tumor.

BACKGROUND AND OBJECTIVE: Phosphenes are visual light phenomena that are experienced when there is no apparent light that stimulates the eye. In oncology, phosphenes are also present during radiation therapy for patients with tumors of the central nervous system, eyes, head and neck. Due to the discomfort of patients treated with irradiation to the head regions, research is conducted to determine whether the dose to the ocular structures is predictive for the occurrence of phosphenes. The objective was to demonstrate the relationship between the doses of the retina and vitreous humour with the appearance of phosphenes. MATERIAL AND METHOD: A descriptive study was carried out in a prospective cohort in 25 patients older than 18 years, with malignant tumours located at the level of the brain, both of primary and secondary origin, subjected to irradiation in 6 MV linear accelerators, during February 2020 to January 2021. As independent variables: Retinal dose and vitreous humour dose, and as dependent variables: Light flashes and stable light. Logistic regression analysis was used for prediction, using the SPSS statistical program (version 26.0). RESULT: A final date of 380 external radiotherapy treatments. The presence of any of the events in a prevalence of 58.7% of the total of fractions. The distribution for the presence of both events, flash of light and stable light, was 69.1%, 20.6% and 10.3% respectively. In the logistic regression analysis, for the light flare, only the dose factor in vitreous was significant (OR: 1.74, IC [1.059-2.419] p: 0.001). For stable light, the dose in the retina (OR: 1.73, IC [1.121-2.341] p: 0.005), and dose in the vitreous humor (OR: 1.82, IC [1.335-2.315] p: 0.003). CONCLUSIONS: There is a predictive relationship between the doses of irradiation of the retina and vitreous humour, for the generation of phosphenes. These results help radiotherapy centres take these anatomical structures into account to reduce the presence of phosphenes in patients. Likewise, it would help to reduce phosphenes, keeping the bunker area illuminated during the treatment.

The interaction of microtubules with stabilizers characterized at biochemical and structural levels.

Since the discovery of paclitaxel and its peculiar mechanism of cytotoxicity, which has made it and its analogues widely used antitumour drugs, great effort has been made to understand the way they produce their effect in microtubules and to find other products that share this effect without the undesired side effects of low solubility and development of multidrug resistance by tumour cells. This chapter reviews the actual knowledge about the biochemical and structural mechanisms of microtubule stabilization by microtubule stabilizing agents, and illustrates the way paclitaxel and its biomimetics induce microtubule assembly, the thermodynamics of their binding, the way they reach their binding site and the conformation they have when bound.

Sodium-selective liquid ion-exchanger microelectrodes for intracellular measurements.

The sodium-selective ligand 1,1,1-tris[1(1)-(2(1)-oxa-4(1)-oxo-5(1)-aza-5(1)-methyl)dodecanyl]propane dissolved in 3-nitro-o-xylene containing a small amount of the lipophilic anion tetrachlorophenyl borate was used as a liquid ion-exchanger in sodium-selective microelectrodes. The microelectrodes gave rapid, stable responses that were linear functions of the logarithm of sodium activity. They were tested under conditions approximating those to be expected in the cell interior, and the results indicated that they can be used to measure intracellular sodium activity without significant interference from intracellular potassium.

Circular dichroism and Fourier transform infrared spectroscopic studies on the secondary structure of Saccharomyces cerevisiae and Escherichia coli phospho enolpyruvate carboxykinases.

The secondary structure of Saccharomyces cerevisiae and Escherichia coli phospho enolpyruvate (PEP) carboxykinases was quantitatively examined using circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopies. From CD analyses, values of 24% alpha-helix and 38% beta-sheet were obtained for the E. coli enzyme, while the corresponding values for the S. cerevisiae PEP carboxykinase were 20% and 36%. Analysis of the amide I' infrared band indicated 20% alpha-helix and 36% beta-sheet for the S. cerevisiae enzyme, while for the E. coli protein values of 40% beta-sheet and between 9 and 36% alpha-helix could be inferred. It is concluded that the bacterial enzyme has more secondary structure elements than the yeast protein. No alteration of the CD or FTIR spectra was detected upon substrate or metal ion binding to any enzyme.

Modulation of cultured brain, adrenal, and aortic endothelial cell glucose transport.

Studies of glucose transporter activity and anti-glucose transporter (GLUT1) immunoblots were performed on different endothelial cell primary cultures (brain capillary, adrenal capillary and aortic) to determine their response to glucose deprivation. Cell cultures were exposed to glucose deprivation (0.5 mM) for 48 h periods and refed (11.0 mM) for 36 additional hours. Control cultures were kept in 11.0 mM glucose for the duration of these studies. Measurements of 2-[3H]deoxy-D-glucose uptake and membrane fraction purification were performed every 12 h during these timecourses. Baseline cytochalasin-B sensitive uptake of 2-deoxy-D-glucose was near three times larger in brain capillary endothelial cells than in adrenal or aortic endothelial cultures. In all three endothelial cell cultures, 2-deoxy-D-glucose uptake increased during glucose deprivation, and returned to control values upon refeeding. Aortic and adrenal cortical endothelia expressed the starvation induced increases 12 h sooner than brain capillary endothelia. Return to control values was also 12 h faster in these cultured endothelia. Immunoblot studies showed that in all three endothelial cell cultures the increases in transporter activity during glucose starvation correlate with increased membrane expression of GLUT1. Quantitative analysis of the anti-GLUT1 immunoblots indicated that induction of GLUT1 following glucose starvation was slower in brain capillary endothelia than in aortic or adrenal endothelia. The slower response by brain capillary endothelial cells may be related to the higher transport rate of glucose in these cells.

Energetics of coupled Na+ and Cl- entry into epithelial cells of bullfrog small intestine.

Na+, K+ and Cl- concentrations (cij) and activities (aij), and mucosal membrane potentials (Em) were measured in epithelial cells of isolated bullfrog (Rana catesbeiana) small intestine. Segments of intestine were stripped of their external muscle layers, and bathed (at 25 degrees C and pH 7.2) in oxygenated Ringer solutions containing 105 mM Na+ and Cl- and 5.4 mM K+. Na+ and K+ concentrations were determined by atomic absorption spectrometry and Cl- concentrations by conductometric titration following extraction of the dried tissue with 0.1 M HNO3. 14C-labelled inulin was used to determine extracellular volume. Em was measured with conventional open tip microelectrodes, aiCl with solid-state Cl-selective silver microelectrodes and aiNa and aiK with Na+ and K+-selective liquid ion-exchanger microelectrodes. The average Em recorded was -34mV. ciNa, ciK and ciCl were 51, 105 and 52 mM. The corresponding values for aiNa, aiK and aiCl were 18, 80 and 33 mM. These results suggest that a large fraction of the cytoplasmic Na+ is 'bound' or sequestered in an osmotically inactive form, that all, or virtually all the cytoplasmic K+ behaves as if in free solution, and that there is probably some binding of cytoplasmic Cl-. aiCl significantly exceeds the level corresponding to electrochemical equilibrium across the mucosal and baso-lateral cell membranes. Earlier studies showed that coupled mucosal entry of Na+ and Cl- is implicated in intracellular Cl- accumulation in this tissue. This study permitted estimation of the steady-state transapical Na+ and Cl- electrochemical potential differences (deltamuNa and deltamuCl). deltamuNa (-7000 J . mol-1; cell minus mucosal medium) was energetically more than sufficient to account for deltamuCl (1000--2000 J . mol-1).

Whole-cell and single channel K+ and Cl- currents in epithelial cells of frog skin.

Whole-cell and single channel currents were studied in cells from frog (R. pipiens and R. catesbiana) skin epithelium, isolated by collagenase and trypsin treatment, and kept in primary cultures up to three days. Whole-cell currents did not exhibit any significant time-dependent kinetics under any ionic conditions used. With an external K gluconate Ringer solution the currents showed slight inward rectification with a reversal potential near zero and an average conductance of 5 nS at reversal. Ionic substitution of the external medium showed that most of the cell conductance was due to K and that very little, if any, Na conductance was present. This confirmed that most cells originate from inner epithelial layers and contain membranes with basolateral properties. At voltages more positive than 20 mV outward currents were larger with K in the medium than with Na or N-methyl-D-glucamine. Such behavior is indicative of a multi-ion transport mechanism. Whole-cell K current was inhibited by external Ba and quinidine. Blockade by Ba was strongly voltage dependent, while that by quinidine was not. In the presence of high external Cl, a component of outward current that was inhibited by the anion channel blocker diphenylamine-2-carboxylate (DPC) appeared in 70% of the cells. This component was strongly outwardly rectifying and reversed at a potential expected for a Cl current. At the single channel level the event most frequently observed in the cell-attached configuration was a K channel with the following characteristics: inward-rectifying I-V relation with a conductance (with 112.5 mM K in the pipette) of 44 pS at the reversal potential, one open and at least two closed states, and open probability that increased with depolarization. Quinidine blocked by binding in the open state and decreasing mean open time. Several observations suggest that this channel is responsible for most of the whole-cell current observed in high external K, and for the K conductance of the basolateral membrane of the intact epithelium. On a few occasions a Cl channel was observed that activated upon excision and brief strong depolarization. The I-V relation exhibited strong outward rectification with a single channel conductance of 48 pS at 0 mV in symmetrical 112 mM Cl solutions. Kinetic analysis showed the presence of two open and at least two closed states. Open time constants and open probability increased markedly with depolarization.(ABSTRACT TRUNCATED AT 400 WORDS)

Reconstruction of protein form with X-ray solution scattering and a genetic algorithm.

We have reconstructed, from experimental approximately 2 nm resolution X-ray solution scattering profiles, the corresponding shapes and sizes of myoglobin, troponin C, spermadhesin PSP-I/PSP-II, chymotrypsinogen A, superoxide dismutase, ovalbumin, tubulin, nitrite reductase, catalase, the structural change of troponin C upon dissociation of the two high affinity Ca(2+), and the solution model structure of a tandem pair of fibronectin type III cytoplasmic domains of integrin alpha6beta4 before determination of its crystal structure. To this purpose we have designed a new genetic algorithm which gradually explores a discrete search space and evolves convergent models made of several hundred beads (down to 0.3 nm radius) best fitting the scattering profile upon Debye calculation, without geometrical constraints or penalty for loose beads. This is a procedure of effective numerical transformation of the one-dimensional scattering profiles into three-dimensional model structures. The number of beads in models is correlated with the protein molecular mass (with one exception). The shape and approximate dimensions of each protein have been retrieved by a set of ten solution models, essentially superimposable with the available crystal structures.

Solution structure of GDP-tubulin double rings to 3 nm resolution and comparison with microtubules.

GDP liganded tubulin, which is inactive in microtubule assembly, polymerizes into rings more readily than the active GTP liganded protein. The structure of double rings made of highly purified GDP-tubulin has been characterized to 3 nm resolution with synchrotron X-ray solution scattering. The scattering profile has characteristic maxima due to closely packed double rings of 38 nm mean diameter, with a 5.5 nm mean spacing between the rings, and a 4.2 nm centre-to-centre spacing between non-globular tubulin monomers within both rings. There are probably 24 and 32 monomers in the inner and outer ring, respectively, and the double ring population is more than 75% homogeneous in size. Comparison of this double ring structure to the lattice of tubulin molecules in microtubules indicates that the tubulin rings are equivalent to pairs of protofilament segments curved tangentially to the microtubule surface, with bending angles of 30 degrees and 22.5 degrees per tubulin alpha beta dimer. When the rings are modelled employing the same non-globular tubulin monomer as in microtubules, the best computer fitted scattering profiles correspond to monomer orientations equivalent to two microtubule protofilaments coiled sideways, with same or opposite polarity. Rings constitute the equilibrium assembly state of GDP-tubulin, which is tensioned inside microtubules after GTP hydrolysis, causing their functional instability. In analogy with other nucleotide binding proteins, the inactive/active structural switch of tubulin is induced by the binding of the gamma phosphate and a coordinated Mg ion. It should involve domain rearrangements which cancel the bending of the tubulin dimer in the ring structure.

Low resolution structure of microtubules in solution. Synchrotron X-ray scattering and electron microscopy of taxol-induced microtubules assembled from purified tubulin in comparison with glycerol and MAP-induced microtubules.

The structure of microtubules has been characterized to 3 nm resolution employing time-resolved X-ray scattering. This has revealed detailed structural features of microtubules not observed before in solution. The polymerization of highly purified tubulin, induced by the antitumour drug taxol, has been employed as a microtubule model system. This assembly reaction requires Mg2+, is optimal at a 1:1 taxol to tubulin heterodimer molar ratio, proceeds with GTP or GDP and is intrinsically reversible. The X-ray scattering profiles are consistent with identical non-globular alpha and beta-tubulin monomers ordered within the known helical surface lattice of microtubules. Purified tubulin-taxol microtubules have a smaller mean diameter (approx. 22 nm) than those induced by microtubule associated proteins or glycerol (approx. 24 nm), but nearly identical wall substructure to the resolution of the measurements. This is because the majority of the former consist of only 12 protofilaments instead of the typical 13 protofilaments, as confirmed by electron microscopy of thin-sectioned, negatively stained and ice-embedded taxol microtubules. It may be concluded that taxol induces a slight reduction of the lateral contact curvature between tubulin monomers. The main fringe pattern observed in cryo-electron micrographs is consistent with a simple 12 protofilament 3-start skewed lattice model. Cylindrical closure of this lattice can be achieved by tilting the lattice 0.8 degrees with respect to the microtubule axis. The closure implies a discontinuity in the type of lateral contacts between the tubulin monomers (regardless of whether these are of the -alpha-beta- or the -alpha-alpha-/-beta-beta- type), which indicates that lateral contacts and the subunit specificity of taxol binding are, to a large degree, equivalent.

An improved method of 90% pancreatectomy using a low dose of streptozotocin at the pancreaticoduodenal artery results in a rapid diabetic stage in dogs.

An improved method of inducing diabetes in dogs was developed. This method included 90% pancreatectomy, 2 mg/kg streptozotocin (STZ) perfused into pancreaticoduodenal artery, and the fixation suture of the duodenum to the costo-abdominal wall. Vasopressin injection administered to the animals before surgery reduced bleeding. All dogs used in this procedure survived and became diabetic. One month after the procedure the pancreatic islets were reduced in volume and the number compared with pancreas tissue obtained during the surgery. Acinar tissue remained with a normal histology, and exocrine function maintained the physiological parameters, except for a soft faecal consistency. We conclude that this procedure is effective in inducing experimental diabetes in dogs.

The effects of intermittent hypoxia training on hematological and aerobic performance in triathletes.

The aim of the present research was to analyze modifications on hematological and aerobic performance parameters after a 7-week intermittent hypoxia training (IHT) program. Eighteen male trained triathletes were divided in two groups: an intermittent hypoxia training group (IHTG: n: 9; 26.0 ± 6.7 years; 173.3 ± 5.9 cm; 66.4 ± 5.9 kg; VO2max: 59.5 ± 5.0 ml/kg/min) that conducted a normoxic training plus an IHT and a control group (CG: n: 9; 29.3 ± 6.8 years; 174.9 ± 4.6 cm; 59.7 ± 6.8 kg; VO2max: 58.9 ± 4.5 ml/kg/min) that performed only a normoxic training. Training process was standardized across the two groups. The IHT program consisted of two 60-min sessions per week at intensities over the anaerobic threshold and atmospheric conditions between 14.5 and 15% FiO2. Before and after the 7-week training, aerobic performance in an incremental running test and hematological parameters were analyzed. After this training program, the IHTG showed higher hemoglobin and erythrocytes (p < 0.05) values than in the CG. In terms of physiological and performance variables, between the two groups no changes were found. The addition of an IHT program to normoxic training caused an improvement in hematological parameters but aerobic performance and physiological variables compared to similar training under normoxic conditions did not increase.

Effect of 6 weeks of whole body vibration training on total and segmental body composition in healthy young adults.

The applied use of new technologies to enhance performance and improve health has been increasing. Initially, whole body vibration training (WBVT) was used as system to improve elite athlete performance. However, this is also used to improve body composition, especially there is a great attention on the effectiveness of WBVT to reduce fat and body weight, with a potential increase in muscle tissue. The aim of this study was to investigate the effects of a 6-week vibration-training program on total and segmental body composition in a group of physically healthy participants. The final study sample included 64 healthy young adults. Subjects were randomly allocated into the control group (CG: n = 26; 16 males and 10 females) and the experimental group (EGWBVT: n = 38; 19 males and 19 females). The program lasted six weeks with a frequency of three sessions per week and each session varied in intensity. There were not found statistically significant differences in any of the body composition variables analysed. This study suggests that a six-week vibration-training program with an increasing intensity (7.2 g-32.6 g) in healthy young adults that are not overweight did not alter total and segmental body composition.

A step toward the prediction of the fluorescence lifetimes of tryptophan residues in proteins based on structural and spectral data.

A method is presented that allows the calculation of the lifetimes of tryptophan residues on the basis of spectral and structural data. It is applied to two different proteins. The calcium binding protein from the sarcoplasm of the muscles of the sand worm Nereis diversicolor (NSCP) changes its conformation upon binding of Ca2+ or Mg2+. NSCP contains three tryptophan residues at position 4, 57, and 170, respectively. The fluorescence lifetimes of W57 are investigated in a mutant in which W4 and W170 have been replaced. The time resolved fluorescence properties of W57 are linked to its different microconformations, which were determined by a molecular dynamics simulation map. Together with the determination of the radiative rate constant from the wavelength of maximum intensity of the decay associated spectra, it was possible to determine an exponential relation between the nonradiative rate constant and the distance between the indole CE3 atom and the carbonyl carbon of the peptide bond reflecting a mechanism of electron transfer as the main determinant of the value for the nonradiative rate constant. This result allows the calculation of the fluorescence lifetimes from the protein structure and the spectra. This method was further tested for the tryptophan of Ha-ras p21 (W32) and for W43 of the Tet repressor, which resulted in acceptable values for the predicted lifetimes.

Characterization of the hinges of the effector loop in the reaction pathway of the activation of ras-proteins. Kinetics of binding of beryllium trifluoride to V29G and I36G mutants of Ha-ras-p21.

This work experimentally confirms the pathway of activation of Ha-ras-p21, which was calculated by the method of Targeted Molecular Dynamics (TMD) (Díaz JF, Wroblowski B, Schlitter J, Engelborghs Y, 1997a, Proteins Struct Funct Genet 28:434-451). The process can be studied experimentally by analyzing the binding of BeF3- to the GDP complex of the active fluorescent mutant Y32W (Díaz JF, Sillen A, Engelborghs Y, 1997b, J Biol Chem 227:23138-23143). Two mutants, V29G and 136G, have been constructed at both sides of the effector loop of the active fluorescent mutant. This was done to check the proposed reaction pathway and to provide further insight into the mechanism of the activation of ras proteins. Both mutations accelerate the conformational isomerization with two orders of magnitude, demonstrating convincingly the role of these residues as hinges of the effector loop in one or more of the transitions of the conformational change. These results provide experimental support to the pathway calculated by TMD analysis.

Role of the switch II region in the conformational transition of activation of Ha-ras-p21.

The role of the switch II region in the conformational transition of activation of Ha-ras-p21 has been investigated by mutating residues predicted to act as hinges for the conformational transition of this loop (Ala59, Gly60, and Gly75) (Díaz JF, Wroblowski B, Schlitter J, Engelborghs Y, 1997, Proteins 28:434-451), as well as mutating the catalytic residue Gln61. The proposed mutations of the hinge residues decrease the rate of the conformational transition of activation as measured by the binding of BeF3- to the GDP-p21 complex. Also, the thermodynamic parameters of the binding reaction are altered by a factor between three and five, depending on the temperature. (Due to changes in activation and reaction enthalpies, partially compensated by entropy changes.) The control mutation Q61H in which only the catalytic residue is changed has only a limited effect on the kinetic rate constants of the conformational transition and on the thermodynamic parameters of the reaction. The fact that mutations of the hinge residues of the switch II region affect both the binding of the phosphate analog and the conformational transition of activation indicates that the switch II is implicated both in the early and the late states of the transition.