Analytical ultracentrifugation (AUC) is a first principles based method to determine absolute sedimentation coefficients and buoyant molar masses of macromolecules and their complexes, reporting on their size and shape in free solution. The purpose of this multi-laboratory study was to establish the precision and accuracy of basic data dimensions in AUC and validate previously proposed calibration techniques. Three kits of AUC cell assemblies containing radial and temperature calibration tools and a bovine serum albumin (BSA) reference sample were shared among 67 laboratories, generating 129 comprehensive data sets. These allowed for an assessment of many parameters of instrument performance, including accuracy of the reported scan time after the start of centrifugation, the accuracy of the temperature calibration, and the accuracy of the radial magnification. The range of sedimentation coefficients obtained for BSA monomer in different instruments and using different optical systems was from 3.655 S to 4.949 S, with a mean and standard deviation of (4.304 ± 0.188) S (4.4%). After the combined application of correction factors derived from the external calibration references for elapsed time, scan velocity, temperature, and radial magnification, the range of s-values was reduced 7-fold with a mean of 4.325 S and a 6-fold reduced standard deviation of ± 0.030 S (0.7%). In addition, the large data set provided an opportunity to determine the instrument-to-instrument variation of the absolute radial positions reported in the scan files, the precision of photometric or refractometric signal magnitudes, and the precision of the calculated apparent molar mass of BSA monomer and the fraction of BSA dimers. These results highlight the necessity and effectiveness of independent calibration of basic AUC data dimensions for reliable quantitative studies.
We describe 5 cases (4 males, 14-43 years old; 1 female, 61 years old) of primary cutaneous T-cell lymphoproliferative lesions expressing a CD8/granzyme/CD30-positive phenotype. Four cases were compatible with lymphomatoid papulosis (LyP) based on the clinical course, which was recurrent asymptomatic papular nodular lesions over years responding to methotrexate; granulomatous inflammation and lack of other inflammatory cell elements were characteristic. In 1 case, an initial erroneous diagnosis was made of aggressive epidermotropic CD8+ T-cell lymphoma. The fifth case in this series was first interpreted as representing primary cutaneous anaplastic large cell lymphoma but was later recategorized as primary cutaneous aggressive epidermotropic CD8+ cytotoxic T-cell lymphoma owing to the extent of extracutaneous dissemination, including testicular involvement and disease progression despite chemotherapeutic intervention. Although all cases of LyP showed sharp cytoplasmic membrane staining with perinuclear Golgi accentuation with CD30, the recategorized case of aggressive epidermotropic CD8 cytotoxic T-cell lymphoma manifested only a weak cytoplasmic staining pattern. CD8 LyP defines a distinctive entity with characteristic light microscopic and phenotypic findings and has a predilection for young males. CD30 expression can occur in other forms of CD8 lymphoproliferative disease unrelated to primary cutaneous anaplastic large cell lymphoma or LyP.
Aminoglycoside nucleotidyltransferase(2'')-Ia is one of the most often detected enzymes in aminoglycoside-resistant bacteria. Despite its prevalence, little biochemical and biophysical work has been reported for this enzyme. In the current study, substrate specificity and temperature dependence of k(cat) are determined by kinetic assays. Dissociation constants and thermodynamic properties of enzyme-substrate complexes are determined by isothermal titration calorimetry, electron paramagnetic resonance, and fluorescence spectroscopy. Kinetic studies show that aminoglycosides with 2'-NH(2) are better substrates (higher k(cat)/K(m)) than ones with 2'-OH when magnesium(II) is used as the catalytically required divalent cation. The activity is reduced 10-fold for substrates with 2'-NH(2) when manganese(II) replaces magnesium as the required metal. However, kanamycin A, which has a 2'-OH, shows a much smaller decrease in activity when manganese substitutes for magnesium as the divalent cation. Temperature dependence studies show the activation energy of catalysis to be 19.2 kcal/mol and the temperature optimum between 30 and 32 degrees C. The binding of the aminoglycoside substrate tobramycin to the enzyme occurs with a favorable enthalpy which compensates for a large entropic penalty to yield a negative DeltaG value for the complex formation. Enthalpy of binding is less exothermic in the presence of metal-nucleotide. However, due to the more favorable entropy, a more favorable DeltaG is observed for the formation of the enzyme-metal-nucleotide:aminoglycoside complex. Tobramycin binds to ANT(2' ') with a dissociation constant of 0.6 microM, which is further reduced by 3-fold when metal-nucleotide is present. Binding of ATP to the enzyme is determined to be very weak in the absence of a divalent cation, and becomes 2 orders of magnitude tighter when magnesium or manganese is present. Binding studies also show that, in addition to binding to the enzyme in the form of metal-nucleotide complex, a second catalytically required metal binds to an additional site on the enzyme.
Thyroid hormones such as 3,3′,5 triiodo-L-thyronine (T3) control numerous aspects of mammalian development and metabolism. The actions of such hormones are mediated by specific thyroid hormone receptors (TRs). TR belongs to the nuclear receptor family of modular transcription factors that binds to specific DNA-response elements within target promoters. These receptors can function as homo-or heterodimers such as TR:9-cis retinoic acid receptor (RXR). Here, we present the atomic resolution structure of the TRα•T3:RXRα•9-cis retinoic acid (9c) ligand binding domain heterodimer complex at 2.95 Å along with T3 hormone binding and dissociation and coactivator binding studies. Our data provide a structural basis for allosteric communication between T3 and 9c and negative cooperativity between their binding pockets. In this structure, both TR and RXR are in the active state conformation for optimal binding to coactivator proteins. However, the structure of TR•T3 within TR•T3:RXR•9c is in a relative state of disorder, and the observed kinetics of binding show that T3 dissociates more rapidly from TR•T3:RXR•9c than from TR•T3:RXR. Also, coactivator binding studies with a steroid receptor coactivator-1 (receptor interaction domains 1-3) fragment show lower affinities (K a ) for TR•T3:RXR•9c than TR•T3:RXR. Our study corroborates previously reported observations from cell-based and binding studies and offers a structural mechanism for the repression of TR•T3:RXR transactivation by RXR agonists. Furthermore, the recent discoveries of multiple endogenous RXR agonists that mediate physiological tasks such as lipid biosynthesis underscore the pharmacological importance of negative cooperativity in ligand binding within TR:RXR heterodimers.allostery | thyroid receptor
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