Reagents for astatination of biomolecules: comparison of the in vivo distribution and stability of some radioiodinated/astatinated benzamidyl and nido-carboranyl compounds

An investigation has been conducted to assess the in vivo stability of a series of astatinated benzamides and astatinated nido-carborane compounds in mice. It was hypothesized that the higher bond strength of boron-astatine bonds in the nido-carboranes might provide increased stability toward in vivo deastatination. Four tri-n-butylstannylbenzamides were prepared for radiohalogenation and evaluation in vivo. Those compounds were N-propyl-4-(tri-n-butylstannyl)benzamide 1a, N-propyl-3-(tri-n-butylstannyl)benzamide 2a, ethyl 4-tri-n-butylstannylhippurate 3a, and 4-tri-n-butylstannyl-hippuric acid 4a. Seven mono-nido-carboranyl derivatives were prepared for radiohalogenation and in vivo evaluation. Four of the seven mono-carboranyl derivatives (5a, 6a, 7a, 13a) contained a 3-(nido-carboranyl)propionamide functionality, and the remaining compounds (8a, 8g, 10a) contained a 4-(nido-carboranyl)aniline functionality. Two additional derivatives (11a, 12a) were prepared that contained bis-(nido-carboranylmethyl)benzene moieties (also referred to as Venus flytrap complexes (VFCs). All benzamide and nido-carborane compounds underwent facile iodination and radiohalogenation, except a 4-(nido-carboranyl)aniline derivative, 8a. Iodination of 8a resulted in a mixture, of which the desired iodinated product was a minor component. Therefore, radiohalogenation was not attempted. It is believed that the mixture of products is due to the presence of a thiourea bond. Previous studies have shown that thiourea bonds can interfere with halogenation reactions. In vivo comparisons of the compounds were conducted by co-injection of dual labeled (125/131I and 211At) compounds. Tissue distribution data were obtained at 1 and 4 h postinjection of the radiolabeled compounds, as that was sufficient to determine if astatine was being released. Stability of the astatinated compound was assessed by the difference in concentration of radioiodine and astatine in lung and spleen. All of the benzamides were found to undergo rapid deastatination in vivo. The nido-carborane derivatives appeared to be slightly more stable to in vivo deastatination; however, they had long blood residence times. The surprising finding was that the VFC derivatives did not release 211At in vivo, even though they rapidly localized to liver. This finding provides encouragement that stable conjugates of 211At may be attained if appropriate modifications of the VFC can be made to redirect their excretion through the renal system.     

Expression and purification of rat recombinant aminopeptidase B secreted from baculovirus-infected insect cells

Aminopeptidase B (Ap-B) is a ubiquitous enzyme and its physiological function still remains an open question. This Zn2+ -exopeptidase catalyzes the amino-terminal cleavage of basic residues of peptide or protein substrates, indicating a role in precursor processing. In addition, the enzyme exhibits a residual capacity to hydrolyze leukotriene A4 (LTA4) into the pro-inflammatory lipid mediator leukotriene B4 (LTB4) in vitro. This potential bi-functional nature of Ap-B is supported by a close structural relationship with LTA4 hydrolase, which hydrolyzes LTA4 into LTB4, in vivo, and exhibits an aminopeptidase activity, in vitro. Structural studies are necessary for the detailed understanding of the bi-functional enzymatic mechanism of Ap-B. In this study, we report cDNA cloning, baculovirus expression, and purification of the rat Ap-B (rAp-B). The Ap-B cDNA was constructed from extracted rat testes total RNA and introduced into the pBAC1 baculovirus transfer vector to generate recombinant baculoviruses. rAp-B expression, with or without COOH-hexahistidine tag, was tested in two different insect cell hosts (Sf9 and H5). The enzyme is secreted into the insect cell culture medium, which allowed a rapid purification of the protein. The His-tagged rAp-B was purified using metal affinity resin while the native recombinant rAp-B was partially purified using a single step DEAE Trisacryl ion exchange column. Although the recombinant rAp-B exhibits biochemical properties equivalent to those of the rat testes purified protein, the presence of the histidine-tag seems to partially inhibit the exopeptidase activity. However, this report shows that baculovirus-infected cells are a useful system to produce rat Ap-B for use in studying enzymatic mechanisms in vitro and 3D structure.     

Label‐free characterization of carbonic anhydrase—novel inhibitor interactions using surface plasmon resonance,isothermal titration calorimetry and fluorescence‐based thermal shift assays

This work describes the development of biophysical unbiased methods to study the interactions between new designed compounds and carbonic anhydrase II (CAII) enzyme. These methods have to permit both a screening of a series of sulfonamide derivatives and the identification of a lead compound after a thorough study of the most promising molecules. Interactions data were collected using surface plasmon resonance (SPR) and thermal shift assay (TSA). In the first step, experiments were performed with bovine CAII isoform and were extended to human CAII. Isothermal titration calorimetry (ITC) experiments were also conducted to obtain thermodynamics parameters necessary for the processing of the TSA data. Results obtained with this reference methodology demonstrate the effectiveness of SPR and TSA. K_D values obtained from SPR data were in perfect accordance with ITC. For TSA, despite the fact that the absolute values of K_D were quite different, the same affinity scale was obtained for all compounds. The binding affinities of the analytes studied vary by more than 50 orders of magnitude; for example, the K_D value determined by SPR were 6 ± 4 and 299 ± 25 nM for compounds 1 and 3, respectively. This paper discusses some of the theoretical and experimental aspects of the affinity‐based methods and evaluates the protein consumption to develop methods for the screening of further new compounds. The double interest of SPR, that is, for screening and for the quick thorough study of the interactions parameters (k_a, k_d, and K_D), leads us to choose this methodology for the study of new potential inhibitors. Copyright © 2013 John Wiley & Sons, Ltd.     

静磁场对单株人体体表正常菌生长影响的研究

本文通过40mT和120mT两种静磁场作用下表皮葡萄球菌生长过程的研究,发现试验所选强度静磁场加速了表皮葡萄球菌在对数生长期的生长速率,而在进入稳定生长期后其生长速率反而低于对照组,但就整个生长周期而言,静磁场作用下表皮葡萄球菌的总量大于对照组,表明了试验所选静磁场对表皮葡萄球菌生长有一定促进作用.     

The efficiency of human cytomegalovirus pp65(495-503) CD8+ T cell epitope generation is determined by the balanced activities of cytosolic and endoplasmic reticulum-resident peptidases

Control of human CMV (HCMV) infection depends on the cytotoxic activity of CD8(+) CTLs. The HCMV phosphoprotein (pp)65 is a major CTL target Ag and pp65(495-503) is an immunodominant CTL epitope in infected HLA-A*0201 individuals. As immunodominance is strongly determined by the surface abundance of the specific epitope, we asked for the components of the cellular Ag processing machinery determining the efficacy of pp65(495-503) generation, in particular, for the proteasome, cytosolic peptidases, and endoplasmic reticulum (ER)-resident peptidases. In vitro Ag processing experiments revealed that standard proteasomes and immunoproteasomes generate the minimal 9-mer peptide epitope as well as N-terminal elongated epitope precursors of different lengths. These peptides are largely degraded by the cytosolic peptidases leucine aminopeptidase and tripeptidyl peptidase II, as evidenced by increased pp65(495-503) epitope presentation after leucine aminopeptidase and tripeptidyl peptidase II knockdown. Additionally, with prolyl oligopeptidase and aminopeptidase B we identified two new Ag processing machinery components, which by destroying the pp65(495-503) epitope limit the availability of the specific peptide pool. In contrast to cytosolic peptidases, silencing of ER aminopeptidases 1 and 2 strongly impaired pp65(495-503)-specific T cell activation, indicating the importance of ER aminopeptidases in pp65(495-503) generation. Thus, cytosolic peptidases primarily interfere with the generation of the pp65(495-503) epitope, whereas ER-resident aminopeptidases enhance such generation. As a consequence, our experiments reveal that the combination of cytosolic and ER-resident peptidase activities strongly shape the pool of specific antigenic peptides and thus modulate MHC class I epitope presentation efficiency.     

Diphasic ventilatory response to hypoxia in newborn lambs

The ventilatory response of newborn lambs to hypoxemia was evaluated in two groups of seven awake lambs studied at 2 and 7 days of life. Minute ventilation (VE) and airway occlusion pressure (P0.1) were monitored as the animals were exposed in sequence to room air, 12% O2 (15 min), 7% O2 (15 min), and room air. On 12 and 7% O2, 2-day-old lambs experienced a brisk hyperventilation followed by a VE depression, previously described in newborns of other species (diphasic response). The 7-day-old lambs had a clear diphasic VE response only on 7% O2 breathing. In the 2-day-old lambs, at the time of the relative VE depression to 12% O2, the respiratory centers showed a persisting responsiveness to further hypoxia; switching to 7% O2 caused a brisk increase in VE and P0.1 of 70 and 130%, respectively, which was followed again by a VE depression. The magnitude of the immediate VE response to hypoxia, taken as an index of the chemoreceptor strength, was inversely related to the magnitude of the VE depression (R = 0.81, P less than 0.001). It was concluded that 1) lambs as well as other neonates have an age-related diphasic VE response to hypoxia; 2) at the time of the VE depression, the respiratory centers maintain their responsiveness to further acute hypoxia; and 3) the weakness of the chemoreceptors in the newborn is a major determinant of the diphasic response.     

Preparation and characterization of anti-tenascin monoclonal antibody-streptavidin conjugates for pretargeting applications.

Radioimmunopretargeting is based on the separate injection of a modified mAb and the radionuclide and most frequently exploits the very high avidity of biotin for streptavidin (SA). Currently, we are evaluating the therapeutic potential of directly labeled monoclonal antibody (mAb) 81C6, reactive with the extracellular matrix protein tenascin, in surgically created glioma resection cavity patients. To be able to investigate pretargeting in this setting, the synthesis of 81C6 mAb-SA conjugates was required. In the current study, we have evaluated five methods for preparing both murine 81C6 (m81C6) and human/mouse chimeric 81C6 (c81C6) SA conjugates with regard to yield, biotin-binding capacity, immunoreactivity, and molecular weight. The 81C6 mAb and SA were coupled by covalent interaction between sulfhydryl groups generated on the mAb via N-succinimidyl-S-acetylthioacetate, dithiothreitol or 2-iminothiolane (2IT), and maleimido-derivatized SA, prepared via sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) or N-succinimidyl-3-(2-pyridyldithio)-propionate. A noncovalent approach involving reaction of a biotinylated mAb, prepared using biotin caproate, and SA also was studied. The evaluation criteria were yield of mAb-SA 215 kDa monomer, as well as conjugate biotin-binding capacity and immunoreactive fraction. The optimal procedure involved activation of m81C6 or c81C6 with 30 equiv of 2IT and reaction of SA with 10 equiv of SMCC and yielded a conjugate with excellent biotin-binding capacity and immunoreactivity. The ((125)I-labeled m81C6)-2IT-SMCC-SA was stable and did not lose biotin-binding capacity after a 72 h incubation in human glioma cyst fluid in vitro. Although the conjugate was stable in murine serum in vivo, its biotin-binding capacity declined rapidly, consistent with high endogenous biotin levels in the mouse. After injection of the radioiodinated conjugate into athymic mice with subcutaneous D-54 MG human glioma xenografts, high tumor uptake (36.0 +/- 10.7% ID/g at 3 days) and excellent tumor:normal tissue ratios were observed.     

Aminopeptidase B (EC 3.4.11.6).

Aminopeptidase B (EC 3.4.11.6) is a Zn(2+)-dependent exopeptidase which selectively removes arginine and/or lysine residues from the NH2-terminus of several peptide substrates including Arg0-Leu-enkephalin, Arg0-Met-enkephalin and Arg-1-Lys0-somatostatin-14. Analysis of its primary structure showed that aminopeptidase-B is structurally related to leukotriene A4 hydrolase, an important enzyme of the arachidonic acid pathway. This structural relationship is further supported by the capacity of aminopeptidase-B to hydrolyse leukotriene A4. Aminopeptidase-B is widely distributed in a number of tissues, including endocrine and non-endocrine cells. Moreover, in rat PC12 pheochromocytoma cells, the enzyme is secreted and associated with the external face of the plasma membrane. Together these data strongly argue in favour of a role of this bi-functional enzyme in the final stages of precursor processing mechanisms occurring either in the secretory pathway, at the plasma membrane, or at both locations.