Substitution of cysteine for selenocysteine in the catalytic center of type III iodothyronine deiodinase reduces catalytic efficiency and alters substrate preference

Human type III iodothyronine deiodinase (D3) catalyzes the conversion of T(4) to rT(3) and of T(3) to 3, 3'-diiodothyronine (T2) by inner-ring deiodination. Like types I and II iodothyronine deiodinases, D3 protein contains selenocysteine (SeC) in the highly conserved core catalytic center at amino acid position 144. To evaluate the contribution of SeC144 to the catalytic properties of D3 enzyme, we generated mutants in which cysteine (D3Cys) or alanine (D3Ala) replaces SeC144 (D3wt). COS cells were transfected with expression vectors encoding D3wt, D3Cys, or D3Ala protein. Kinetic analysis was performed on homogenates with dithiothreitol as reducing cofactor. The Michaelis constant of T(3) was 5-fold higher for D3Cys than for D3wt protein. In contrast, the Michaelis constant of T(4) increased 100-fold. The D3Ala protein was enzymatically inactive. Semiquantitative immunoblotting of homogenates with a D3 antiserum revealed that about 50-fold higher amounts of D3Cys and D3Ala protein are expressed relative to D3wt protein. The relative substrate turnover number of D3Cys is 2-fold reduced for T(3) and 6-fold reduced for T(4) deiodination, compared with D3wt enzyme. Studies in intact COS cells expressing D3wt or D3Cys showed that the D3Cys enzyme is also active under in situ conditions. In conclusion, the SeC residue in the catalytic center of D3 is essential for efficient inner-ring deiodination of T(3) and in particular T(4) at physiological substrate concentrations

Substitution of cysteine for a conserved alanine residue in the catalytic center of type II iodothyronine deiodinase alters interaction with reducing cofactor

Human type II iodothyronine deiodinase (D2) catalyzes the activation of T(4) to T(3). The D2 enzyme, like the type I (D1) and type III (D3) deiodinases, contains a selenocysteine (SeC) residue (residue 133 in D2) in the highly conserved catalytic center. Remarkably, all of the D2 proteins cloned so far have an alanine two residue-amino terminal to the SeC, whereas all D1 and D3 proteins contain a cysteine at this position. A cysteine residue in the catalytic center could assist in enzymatic action by providing a nucleophilic sulfide or by participating in redox reactions with a cofactor or enzyme residues. We have investigated whether D2 mutants with a cysteine (A131C) or serine (A131S) two-residue amino terminal to the SeC are enzymatically active and have characterized these mutants with regard to substrate affinity, reducing cofactor interaction and inhibitor profile. COS cells were transfected with expression vectors encoding wild-type (wt) D2, D2 A131C, or D2 A131S proteins. Kinetic analysis was performed on homogenates with dithiothreitol (DTT) as reducing cofactor. The D2 A131C and A131S mutants displayed similar Michaelis-Menten constant values for T(4) (5 nM) and reverse T(3) (9 nM) as the wt D2 enzyme. The limiting Michaelis-Menten constant for DTT of the D2 A131C enzyme was 3-fold lower than that of the wt D2 enzyme. The wt and mutant D2 enzymes are essentially insensitive to propylthiouracil [concentration inhibiting 50% of activity (IC(50)) > 2 mM] in the presence of 20 mM DTT, but when tested in the presence of 0.2 mM DTT the IC(50) value for propylthiouracil is reduced to about 0.1 mM. During incubations of intact COS cells expressing wt D2, D2 A131C, or D2 A131S, addition of increasing amounts of unlabeled T(4) resulted in the saturation of [(125)I]T(4) deiodination, as reflected in a decrease of [(125)I]T(3) release into the medium. Saturation first appeared at medium T(4) concentrations between 1 and 10 nM. In conclusion: substitution of cysteine for a conserved alanine residue in the catalytic center of the D2 protein does not inactivate the enzyme in vitro and in situ, but rather improves the interaction with the reducing cofactor DTT in vitro

The vulnerable microcirculation in the critically ill pediatric patient

In neonates, cardiovascular system development does not stop after the transition from intra-uterine to extra-uterine life and is not limited to the macrocirculation. The microcirculation (MC), which is essential for oxygen, nutrient, and drug delivery to tissues and cells, also develops. Developmental changes in the microcirculatory structure continue to occur during the initial weeks of life in healthy neonates. The physiologic hallmarks of neonates and developing children make them particularly vulnerable during critical illness; however, the cardiovascular monitoring possibilities are limited compared with critically ill adult patients. Therefore, the development of non-invasive methods for monitoring the MC is necessary in pediatric critical care for early identification of impending deterioration and to enable the initiation and titration of therapy to ensure cell survival. To date, the MC may be non-invasively monitored at the bedside using hand-held videomicroscopy, which provides useful information regarding the microcirculation. There is an increasing number of studies on the MC in neonates and pediatric patients; however, additional steps are necessary to transition MC monitoring from bench to bedside. The recently introduced concept of hemodynamic coherence describes the relationship between changes in the MC and macrocirculation. The loss of hemodynamic coherence may result in a depressed MC despite an improvement in the macrocirculation, which represents a condition associated with adverse outcomes. In the pediatric intensive care unit, the concept of hemodynamic coherence may function as a framework to develop microcirculatory measurements towards implementation in daily clinical practice

Toxicity Profiles In Vivo in Mice and Antitumour Activity in Tumour-Bearing Mice of Di- and Triorganotin Compounds

The in vivo toxicity profiles in mice and the antitumour activity in tumour bearing mice were screened for four di-n-butyltin and five triorganotin carboxylates, di-n-butyltin diterebate (5), bis(phenylacetate) (6), bis(deoxycholate) (7), bis(lithocholate) (8), tri-n-butyltin terebate (9), cinnamate (10), and triphenyltin terebate (11)

Accuracy and the influence of marrow fat on quantitative CT and dual-energy X-ray absorptiometry measurements of the femoral neck in vitro

Abstract Bone mineral measurements with quantitative computed tomography (QCT) and dual-energy X-ray absorptiometry (DXA) were compared with chemical analysis (ChA) to determine (1) the accuracy and (2) the influence of bone marrow fat. Total bone mass of 19 human femoral necks in vitro was determined with QCT and DXA before and after defatting. ChA consisted of defatting and decalcification of the femoral neck samples for determination of bone mineral mass (BmM) and amount of fat. The mean BmM was 4.49 g. Mean fat percentage was 37.2% (23.3%–48.5%). QCT, DXA and ChA before and after defatting were all highly correlated (r>0.96,p<0.0001). Before defatting the QCT values were on average 0.35 g less than BmM and the DXA values were on average 0.65 g less than BmM. After defatting, all bone mass values increased; QCT values were on average 0.30 g more than BmM and DXA values were 0.29 g less than BmM. It is concluded that bone mineral measurements of the femoral neck with QCT and DXA are highly correlated with the chemically determined bone mineral mass and that both techniques are influenced by the femoral fat content

Endoanal MRI of the anal sphincter complex: correlation with cross-sectional anatomy and histology

The purpose of this study was to correlate the in vivo endoanal MRI findings of the anal sphincter with the cross-sectional anatomy and histology. Fourteen patients with rectal tumours were examined with a rigid endoanal MR coil before undergoing abdominoperineal resection. In addition, 12 cadavers were used to obtain cross-sectional anatomical sections. The images were correlated with the histology and anatomy of the resected rectal specimens as well as with the cross-sectional anatomical sections of the 12 cadavers. The findings in 8 patients, 11 rectal preparations, and 10 cadavers, could be compared. In these cases, there was an excellent correlation between endoanal MRI and the cross-sectional cadaver anatomy and histology. With endoanal MRI, all muscle layers of the anal canal wall, comprising the internal anal sphincter, longitudinal muscle, the external anal sphincter and the puborectalis muscle wer