Evaluation of maleimide derivative of DOTA for site-specific labeling of recombinant affibody molecules

Affibody molecules are a new class of small (7 kDa) scaffold affinity proteins, which demonstrate promising properties as agents for in vivo radionuclide targeting. The Affibody scaffold is cysteine-free and therefore independent of disulfide bonds. Thus, a single thiol group can be engineered into the protein by introduction of one cysteine. Coupling of thiol-reactive bifunctional chelators can enable site-specific labeling of recombinantly produced Affibody molecules. In this study, the use of 1,4,7,10-tetraazacyclododecane-1,4,7-tris-acetic acid-10-maleimidoethylacetamide (MMA-DOTA) for 111 In-labeling of anti-HER2 Affibody molecules His 6-Z HER2:342-Cys and Z HER2:2395-Cys has been evaluated. The introduction of a cysteine residue did not affect the affinity of the proteins, which was 29 pM for His 6-Z HER2:342-Cys and 27 pM for Z HER2:2395-Cys, comparable with 22 pM for the parental Z HER2:342. MMA-DOTA was conjugated to DTT-reduced Affibody molecules with a coupling efficiency of 93% using a 1:1 molar ratio of chelator to protein. The conjugates were labeled with 111 In to a specific radioactivity of up to 7 GBq/mmol, with preserved binding for the target HER2. In vivo, the non-His-tagged variant 111 In-[MMA-DOTA-Cys61]-Z HER2:2395-Cys demonstrated appreciably lower liver uptake than its His-tag-containing counterpart. In mice bearing HER2-expressing LS174T xenografts, 111 In-[MMA-DOTA-Cys61]-Z HER2:2395-Cys showed specific and rapid tumor localization, and rapid clearance from blood and nonspecific compartments, leading to a tumor-to-blood-ratio of 18 +/- 8 already 1 h p.i. Four hours p.i., the tumor-to-blood ratio was 138 +/- 8. Xenografts were clearly visualized already 1 h p.i.     

A panel of 13 region-specific radioimmunoassays for measurements of human chromogranin B

INTRODUCTION: The primary structure of human chromogranin B (CgB) contains 15 pairs of basic amino acids, which are potential cleavage sites for specific endogenous proteases, but also other sites in the molecule can be subjected to cleavage. Several CgB-related peptides have been identified in tissue extracts. MATERIALS AND METHODS: Peptides homologous to defined parts of the human CgB molecule were selected and synthesized. Antibodies were raised and 13 specific radioimmunoassays were developed. Plasma samples from 19 patients with neuroendocrine tumors were collected and measured in all assays. RESULTS: All region-specific assays measured circulating levels of CgB-related peptides. Only five of the assays measured high concentrations of circulating CgB and two of them correlated with that of intact chromogranin A (CgA). CONCLUSION: The assays presented allow measurements of defined regions of CgB and will thus become important tools for further studies of the processing of CgB. One of the assays merit further investigations as a new marker for neuroendocrine tumors.     

Vesicle-mediated export and assembly of pore-forming oligomers of the enterobacterial ClyA cytotoxin

The ClyA protein is a pore-forming cytotoxin expressed by Escherichia coli and some other enterobacteria. It confers cytotoxic activity toward mammalian cells, but it has remained unknown how ClyA is surface exposed and exported from bacterial cells. Outer-membrane vesicles (OMVs) released from the bacteria were shown to contain ClyA protein. ClyA formed oligomeric pore assemblies in the OMVs, and the cytotoxic activity toward mammalian cells was considerably higher than that of ClyA protein purified from the bacterial periplasm. The redox status of ClyA correlated with its ability to form the oligomeric pore assemblies. In bacterial cells with a defective periplasmic disulphide oxidoreductase system, the ClyA protein was phenotypically expressed in a constitutive manner. The results define a vesicle-mediated transport mechanism in bacteria, and our findings show that the localization of proteins to OMVs directly may contribute to the activation and delivery of pathogenic effector proteins.     

Fluorescence polarization as an analytical tool to evaluate galectin-ligand interactions

Galectins are a family of beta-galactose binding lectins associated with functions such as immunological and malignant events. To study the binding affinity of galectins for natural and artificial saccharides and glycoconjugates we have developed an assay using fluorescence polarization. A collection of fluorescein-conjugated saccharides was synthesized and used as probes with galectins-1 and -3 and the two carbohydrate recognition domains of galectin-4. Direct binding of a fixed probe amount with different amounts of each galectin defined specificity and selectivity and permitted selection of the optimal probe for inhibition studies. Then fixed amounts of galectin and selected probe were used to screen the inhibitory potency of a library of nonfluorescent compounds. As the assay is in solution and does not require separation of free and bound probe, it is simple and rapid and can easily be applied to different unlabeled galectins. As all interaction components are known, K(d) values for galectin-inhibitor interaction can be directly calculated without approximation other than the assumption of a simple one-site competition.     

R-Ras contains a proline-rich site that binds to SH3 domains and is required for integrin activation by R-Ras

R-Ras contains a proline-rich motif that resembles SH3 domain-binding sites but that has escaped notice previously. We show here that this site in R-Ras is capable of binding SH3 domains and that the SH3 domain binding may be important for R-Ras function. A fusion protein containing the SH3 domains of the adaptor protein Nck interacted strongly with the R-Ras proline-rich sequence and with the intact protein. The binding was independent of whether R-Ras was in its GDP or GTP form. The Nck binding, which was mediated by the second of the three SH3 domains of Nck, was obliterated by mutations in the proline-rich sequence of R-Ras. The interaction of Nck with R-Ras could also be shown in yeast two-hybrid assays and by co-immunoprecipitation in human cells transfected with Nck and R-Ras. Previous results have shown that the expression of a constitutively active R-Ras mutant, R-Ras(38V), converts mouse 32D monocytic cells into highly adherent cells. Introducing the proline mutations into R-Ras(38V) suppressed the effect of R-Ras on 32D cell adhesion while not affecting GTP binding. These results reveal an unexpected regulatory pathway that controls R-Ras through an SH3 domain interaction. This pathway appears to be important for the ability of R-Ras to control cell adhesion.     

Potential phosphorus release from catch crop shoots and roots after freezing-thawing

Background and aims

Catch crops used for mitigating nutrient losses to water can release phosphorus (P) when exposed to repeated freezing-thawing cycles (FTCs). This study sought to evaluate potential P losses from shoots and roots of eight catch crops.

Methods

Shoots and roots sampled from perennial ryegrass (Lolium perenne L.), cocksfoot (Dactylis glomerata L.), chicory (Cichorium intybus L.), phacelia (Phacelia tanacetifolia L.), red clover (Trifolium pratense L.), white mustard (Sinapis alba L.), oilseed radish (Raphanus sativus var. oleiformis L.) and white radish (R. sativus var. longipinnatus L.) were treated with no freezing, one single FTC, four continuous FTCs and four discontinuous FTCs. All samples were analysed for water-extractable P (WEP), and root samples also for characteristics such as specific root surface area (SSA).

Results

Freezing-thawing significantly increased potential P losses from both shoots and roots compared with no freezing. The two radish species and white mustard contained significantly higher concentrations of WEP than the other species, among which chicory and phacelia had the lowest WEP. On average, shoots had 43 % higher WEP than roots. Cumulative P release from shoots and roots was strongly correlated with their total-P content (p?=?0.006 and p?=?0.002, respectively). Cumulative release of P from taproots was correlated with SSA (p?=?0.03).

Conclusions

Chicory, and possibly phacelia, appear to be promising catch crops for P.     

Expression of 15-lipoxygenase type-1 in human mast cells

Mast cells play a key role in the pathophysiology of asthma. These cells exert their effector functions by releasing a variety of proinflammatory and immunoregulatory compounds. Mast cells infiltrate the bronchial epithelium and smooth muscle to a higher degree in patients with asthma compared to control subjects. 15-Lipoxygenase type-1 (15-LO-1) is a prooxidant enzyme which is expressed in asthmatic lungs leading to formation of pro- and anti-inflammatory mediators. Here we report that interleukin-4 (IL-4) induced the expression of 15-LO-1 in human cord blood derived mast cells (CBMC) as demonstrated by RT-PCR, western blot and immunocytochemistry. The major metabolite of arachidonic acid formed via the 15-LO pathway in IL-4 treated CBMC was identified as 15-ketoeicosatetraenoic acid (15-KETE, also named 15-oxo-ETE) with smaller amounts of 15-hydroxyeicosatetraenoic acid (15-HETE) as identified by HPLC and mass spectrometry (MS/MS). Furthermore, immunohistochemical stainings demonstrated the expression of 15-LO-1 in mast cells in lung and skin in vivo. Osmotic activation of CBMC with mannitol resulted in activation of the 15-LO-1 pathway. In conclusion, the expression of 15-LO-1 and release of 15-LO-1 derived products by mast cells may contribute to the role of these cells in asthma and other inflammatory diseases.     

Generation of Neutralizing Antibodies and Divergence of SIVmac239 in Cynomolgus Macaques Following Short-Term Early Antiretroviral Therapy

Neutralizing antibodies (NAb) able to react to heterologous viruses are generated during natural HIV-1 infection in some individuals. Further knowledge is required in order to understand the factors contributing to induction of cross-reactive NAb responses. Here a well-established model of experimental pathogenic infection in cynomolgus macaques, which reproduces long-lasting HIV-1 infection, was used to study the NAb response as well as the viral evolution of the highly neutralization-resistant SIVmac239. Twelve animals were infected intravenously with SIVmac239. Antiretroviral therapy (ART) was initiated ten days post-inoculation and administered daily for four months. Viral load, CD4⁺ T-cell counts, total IgG levels, and breadth as well as strength of NAb in plasma were compared simultaneously over 14 months. In addition, envs from plasma samples were sequenced at three time points in all animals in order to assess viral evolution. We report here that seven of the 12 animals controlled viremia to below 10⁴ copies/ml of plasma after discontinuation of ART and that this control was associated with a low level of evolutionary divergence. Macaques that controlled viral load developed broader NAb responses early on. Furthermore, escape mutations, such as V67M and R751G, were identified in virus sequenced from all animals with uncontrolled viremia. Bayesian estimation of ancestral population genetic diversity (PGD) showed an increase in this value in non-controlling or transient-controlling animals during the first 5.5 months of infection, in contrast to virus-controlling animals. Similarly, non- or transient controllers displayed more positively-selected amino-acid substitutions. An early increase in PGD, resulting in the generation of positively-selected amino-acid substitutions, greater divergence and relative high viral load after ART withdrawal, may have contributed to the generation of potent NAb in several animals after SIVmac239 infection. However, early broad NAb responses correlated with relatively preserved CD4⁺ T-cell numbers, low viral load and limited viral divergence.