Synthesis and testing of a binary catalytic system for imaging of signal amplification in vivo

We report a binary targeted enzymatic system that is composed of two covalent monoclonal antibody conjugates for specific labeling of cellular targets in vivo. The system utilizes low-molecular weight peroxidase-reducing substrates synthesized by linking 5-hydroxytryptamine (serotonin) with DTPA (5HT-DTPA) for magnetic resonance and radionuclide imaging or with Cy5.5 for near-infrared optical imaging. Initially, the conjugation reaction conditions were optimized to achieve a low level of antiepidermal growth factor receptor (EGFR) antibody (EMD 72000) modification with the N-hydroxysuccinimide ester of 4-hydrazinonicotinate acetone hydrazone (SANH), yielding mAb-HNH conjugate. The resultant modified antibodies were incubated with the periodate-oxidized peroxidase (HRP) or 4-formylbenzoyl-conjugated glucose oxidase (GO), followed by the purification of the resultant mAb-enzyme conjugates by size-exclusion HPLC. The conjugates were further characterized by electrophoresis and were tested by cross-titration on A431 EGFR+ squamous carcinoma or SW620 adenocarcinoma cells (negative control). The conjugates at the optimized concentration ratios were further tested using near-infrared fluorescence microscopy in the presence of Cy5.5 monocarboxy-5-hydroxytryptamide. Further in vitro experiments demonstrated that (1) antibody binding was specific and could be inhibited by free antibody; (2) both antibody conjugates exhibited high enzymatic activity after the binding to the cells; (3) 111In-labeled 5-HT-DTPA was avidly binding to EGFR-positive cells only if both HRP- and GO-conjugates were bound to the cells. The conjugates were tested in vivo using a SPECT imaging experiment, which demonstrated the accumulation of 111In-labeled 5-HT-DTPA substrate at the site containing both conjugates.     

The role of the functional sites of the merlin tumor suppressor in <Emphasis Type="Italic">Drosophila Spermatogenesis</Emphasis>

The Merlin gene of Drosophila is homologous to the human Neurofibromatosis 2 (NF2) gene, an important regulator of proliferation and endocytosis of cell receptors. It was earlier shown that the Thr⁵⁵⁹ residue of the Drosophila Merlin protein was homologous to Ser⁵¹⁸ of the human protein (which was already known to undergo phosphorylation); hence, it was assumed that Thr⁵⁵⁹ of Drosophila also was a substrate of phosphorylation. The mutant Merlin proteins MerT^559D (an analog of the phosphorylated form) and MerT^559A (a nonphosphorylated form) were constructed and tested, under the conditions of ectopic expression, for the ability to correct the spermatogenesis defects induced by the Mer4 mutation. The mutant form MerT^559D was demonstrated to restore the abnormal nebenkern phenotype induced by this mutation, whereas the MerT^559A substituted form did not restore this phenotype. Ectopic expression o the wild-type Merlin protein, MerT^559A mutant form, and mycMer^345–635 truncated protein in a normal genotype resulted in the abnormal nebenkern phenotype, whereas this phenotype was not observed in the case of ectopic expression of the Mer^T559D analog of the phosphorylated form. Ectopic expression of the mycMer³, mycMer^ΔBB, and mycMer^1–379 truncate variants led to disturbance of meiotic cytokinesis.     

Study of glycosylation with N-trichloroacetyl-D-glucosamine derivatives in the syntheses of the spacer-armed pentasaccharides sialyl lacto-N-neotetraose and sialyl lacto-N-tetraose, their fragments, and analogues.

The syntheses of 2-aminoethyl glycosides of the pentasaccharides Neu5Ac-alpha(2-->3)-Gal-beta(1-->4)-GlcNAc-beta(1-->3)-Gal-beta(1-->4)-Glc and Neu5Ac-alpha(2-->3)-Gal-beta(1-->3)-GlcNAc-beta(1-->3)-Gal-beta(1-->4)-Glc, their asialo di-, tri-, and tetrasaccharide fragments, and analogues included a systematic study of glycosylation with variously protected mono- and disaccharide donors derived from N-trichloroacetyl-D-glucosamine of galactose, lactose, and lactosamine glycosyl acceptors bearing benzoyl protection around the OH group to be glycosylated. Despite the low reactivity of these acceptors, stereospecificity and good to excellent yields were obtained with NIS-TfOH-activated thioglycoside donors of such type, or with AgOTf-activated glycosyl bromides, while other promotors, as well as a trichloroacetimidate donor, were less effective, and a beta-acetate donor was inactive. In NIS-TfOH-promoted glycosylation with the thioglycosides, the use of TfOH in catalytic amount led to rapid formation of the corresponding oxazoline, but the quantity of TfOH necessary for further efficient coupling with an acceptor depended on the reactivity of the donor, varying from 0.07 equiv for a 3,6-di-O-benzylated monosaccharide derivative to 2.1 equiv for a peracetylated disaccharide one. In the glycosylation products, the N-trichloroacetyl group was easily converted into N-acetyl by alkaline hydrolysis followed by N-acetylation.     

The role of Drosophila Merlin in spermatogenesis

Background

Drosophila Merlin, the homolog of the human Neurofibromatosis 2 (NF2) gene, is important for the regulation of cell proliferation and receptor endocytosis. Male flies carrying a Mer ³ allele, a missense mutation (Met¹⁷⁷→Ile) in the Merlin gene, are viable but sterile; however, the cause of sterility is unknown.

Results

Testis examination reveals that hemizygous Mer ³ mutant males have small seminal vesicles that contain only a few immotile sperm. By cytological and electron microscopy analyses of the Mer ³, Mer ⁴ (Gln¹⁷⁰→stop), and control testes at various stages of spermatogenesis, we show that Merlin mutations affect meiotic cytokinesis of spermatocytes, cyst polarization and nuclear shaping during spermatid elongation, and spermatid individualization. We also demonstrate that the lethality and sterility phenotype of the Mer ⁴ mutant is rescued by the introduction of a wild-type Merlin gene. Immunostaining demonstrates that the Merlin protein is redistributed to the area associated with the microtubules of the central spindle in telophase and its staining is less in the region of the contractile ring during meiotic cytokinesis. At the onion stage, Merlin is concentrated in the Nebenkern of spermatids, and this mitochondrial localization is maintained throughout sperm formation. Also, Merlin exhibits punctate staining in the acrosomal region of mature sperm.

Conclusion

Merlin mutations affect spermatogenesis at multiple stages. The Merlin protein is dynamically redistributed during meiosis of spermatocytes and is concentrated in the Nebenkern of spermatids. Our results demonstrated for the first time the mitochondrial localization of Merlin and suggest that Merlin may play a role in mitochondria formation and function during spermatogenesis.     

The presence of water improves reductive openings of benzylidene acetals with trimethylaminoborane and aluminium chloride

The acidic reagent formed in situ from anhydrous AlCl(3) and H(2)O in 3:1 ratio is much more efficient for the reductive openings of the cyclic benzylidene acetals with Me(3)N x BH(3) in tetrahydrofurane than the AlCl(3) alone. Under proposed conditions, the dioxane-type 4,6-O-bezylidene acetals of hexopyranosides give regioselectively the corresponding 4-hydroxy,6-O-benzyl derivatives in excellent yields. Reductive openings of the dioxolane-type 3,4-O-benzylidene acetals of galactopyranoside are also very efficient and regioselective and give either 3-O-benzyl derivative (from 3,4-O-exo-benzylidene acetal) or 4-O-benzyl derivative (from 3,4-O-endo-benzylidene acetal) depending on the configuration of the acetal carbon atom.     

Novel antibacterial proteins from entomocidal crystals of Bacillus thuringiensis ssp. israelensis

Proteins with molecular masses of 36 and 34 kDa (Bti36 and Bti34) were isolated from entomocidal crystals formed by Bacillus thuringiensis ssp. israelensis cells. The samples of Bti36 contained the admixture of a protein with a molecular mass of 33 kDa (Bti33), apparently a product of proteolysis of Bti36. These 3 proteins are significantly different in N-terminal sequences from known delta-endotoxins of B. thuringiensis and show antibacterial activity toward Micrococcus luteus. The combination of Bti36 and Bti33 also suppresses the growth of some other microorganisms including Streptomyces chrysomallus. The effects of the mixture of Bti36 and Bti33 on the M. luteus cell surface and on the surface of S. chrysomallus cells and exospores are similar, but they are different from the effect of endotoxin Cry11A on micrococcal cells.     

DELAYED URIC ACID ACCUMULATION IN PLASMA PROVIDES ADDITIONAL ANTI-OXIDANT PROTECTION AGAINST IRON-TRIGGERED OXIDATIVE STRESS AFTER A WINGATE TEST

Reactive oxygen species are produced during anaerobic exercise mostly by Fe ions released into plasma and endothelial/muscle xanthine oxidase activation that generates uric acid (UA) as the endpoint metabolite. Paradoxically, UA is considered a major antioxidant by virtue of being able to chelate pro-oxidative iron ions. This work aimed to evaluate the relationship between UA and plasma markers of oxidative stress following the exhaustive Wingate test. Plasma samples of 17 male undergraduate students were collected before, 5 and 60 min after maximal anaerobic effort for the measurement of total iron, haem iron, UA, ferric-reducing antioxidant activity in plasma (FRAP), and malondialdehyde (MDA, biomarker of lipoperoxidation). Iron and FRAP showed similar kinetics in plasma, demonstrating an adequate pro-/antioxidant balance immediately after exercise and during the recovery period (5–60 min). Slight variations of haem iron concentrations did not support a relevant contribution of rhabdomyolysis or haemolysis for iron overload following exercise. UA concentration did not vary immediately after exercise but rather increased 29% during the recovery period. Unaltered MDA levels were concomitantly measured. We propose that delayed UA accumulation in plasma is an auxiliary antioxidant response to post-exercise (iron-mediated) oxidative stress, and the high correlation between total UA and FRAP in plasma (R-Square = 0.636; p = 0.00582) supports this hypothesis.     

Seasonal Dynamics of the Chlorophyll and Carotenoid Content in the Leaves of Steppe and Forest Plants on Species and Community Level

Russian Journal of Plant Physiology - The content of chlorophylls (Сhl) a and b and carotenoids (Сar) during the growing season was studied in the leaves of 31 species of steppe and...     

Rapid Structural Characterization of Human Antibody-Antigen Complexes through Experimentally Validated Computational Docking

If we understand the structural rules governing antibody (Ab)-antigen (Ag) interactions in a given virus, then we have the molecular basis to attempt to design and synthesize new epitopes to be used as vaccines or optimize the antibodies themselves for passive immunization. Comparing the binding of several different antibodies to related Ags should also further our understanding of general principles of recognition.To obtain and compare the three-dimensional structure of a large number of different complexes, however, we need a faster method than traditional experimental techniques. While biocomputational docking is fast, its results might not be accurate. Combining experimental validation with computational prediction may be a solution.As a proof of concept, here we isolated a monoclonal Ab from the blood of a human donor recovered from dengue virus infection, characterized its immunological properties, and identified its epitope on domain III of dengue virus E protein through simple and rapid NMR chemical shift mapping experiments. We then obtained the three-dimensional structure of the Ab/Ag complex by computational docking, using the NMR data to drive and validate the results. In an attempt to represent the multiple conformations available to flexible Ab loops, we docked several different starting models and present the result as an ensemble of models equally agreeing with the experimental data. The Ab was shown to bind a region accessible only in part on the viral surface, explaining why it cannot effectively neutralize the virus.