In vivo evaluation of cysteine-based chelators for attachment of 99mTc to tumor-targeting Affibody molecules

Affibody molecules present a new class of affinity proteins, which utilizes a scaffold based on a 58-amino acid domain derived from protein A. The small (7 kDa) Affibody molecule can be selected to bind to cell-surface targets with high affinity. An Affibody molecule (ZHER2:342) with a dissociation constant (Kd) of 22 pM for binding to the HER2 receptor has been reported earlier. Preclinical and pilot clinical studies have demonstrated the utility of radiolabeled ZHER2:342 in imaging of HER2-expressing tumors. The small size and cysteine-free structure of Affibody molecules enable complete peptide synthesis and direct incorporation of radionuclide chelators. The goal of this study was to evaluate if incorporation of the natural peptide sequences cysteine-diglycine (CGG) and cysteine-triglycine (CGGG) sequences would enable labeling of Affibody molecules with 99mTc. In a model monomeric form, the chelating sequences were incorporated by peptide synthesis. The HER2-binding affinity was 280 and 250 pM for CGG-ZHER2:342 and CGGG-ZHER2:342, respectively. Conjugates were directly labeled with 99mTc with 90% efficiency and preserved the capacity to bind specifically to HER2-expressing cells. The biodistribution in normal mice showed a rapid clearance from the blood and the majority of organs (except kidneys). In the mice bearing SKOV-3 xenografts, tumor uptake of 99mTc-CGG-ZHER2:342 was HER2-specific and a tumor-to-blood ratio of 9.2 was obtained at 6 h postinjection. Gamma-camera imaging with 99mTc-CGG-ZHER2:342 clearly visualized tumors at 6 h postinjection. The results show that the use of a cysteine-based chelator enables 99mTc-labeling of Affibody molecules for imaging.     

Effects of cyanobacterial extracellular products and gibberellic acid on salinity tolerance in Oryza sativa L

The XI International Rotifer Symposium was held during 11–18 March, 2006 at the National Autonomous University of Mexico Campus Iztacala located at the North Mexico City (Mexico). These triennial international meetings, first organized in Austria by Late Ruttner-Kolisko in September 1976, are gradually becoming the focal point of discussion and collaboration from rotifer workers across the world. The present XI symposium was attended by 125 participants from 20 nations. During this meeting, different themes of rotifer research from morphology to molecular biology were considered. In addition, there were four invited lectures and four workshops covering different themes of the symposium. During the last 30 years, rotifer research has witnessed gradual shift from the conventional morphological taxonomy to molecular and evolutionary systematics. While the basic rotifer ecological studies continue today, applied areas such as ecotoxicology and aquaculture have taken key roles in the recent meetings. The international rotifer meetings provide ample opportunities not only for exchange of ideas and recent research, but also for material and in establishing inter-personal relationships. Over the last 30 years, the number of participants attending the rotifer meetings has increased.     

Studies of esterase 6 in Drosophila melanogaster. XVIII. Biochemical differences between the slow and fast allozymes

Most natural populations of Drosophila melanogaster are polymorphic for two major electrophoretic variants at the esterase-6 locus. The frequency of the EST 6F allozyme is greatest in populations in warmer latitudes, whereas the EST 6S allozyme is predominant in colder latitudes. Latitudinal clines in electromorph frequencies are found on three continents. Purified preparations of the allozymes have been characterized for their pH optimum, substrate specificity, organophosphate inhibition, alcohol activation, thermal stability, and kinetic parameters. These and previous analyses of the EST 6 allozymes reveal that the two variants have differences in their physical and kinetic properties that may provide a basis for the selective maintenance of the polymorphisms and an explanation of the clinal variation observed in natural populations.      

Higher number of <Emphasis Type="Italic">Helicobacter pylori</Emphasis> CagA EPIYA C phosphorylation sites increases the risk of gastric cancer,but not duodenal ulcer

Background  

Helicobacter pylori infection is one of the most common infections worldwide and is associated with gastric cancer and peptic ulcer. Bacterial virulence factors such as CagA have been shown to increase the risk of both diseases. Studies have suggested a causal role for CagA EPIYA polymorphisms in gastric carcinogenesis, and it has been shown to be geographically diverse. We studied associations between H. pylori CagA EPIYA patterns and gastric cancer and duodenal ulcer, in an ethnically admixed Western population from Brazil. CagA EPIYA was determined by PCR and confirmed by sequencing. A total of 436 patients were included, being 188 with gastric cancer, 112 with duodenal ulcer and 136 with gastritis.     

A whole genome screen for HIV restriction factors

Background

Human T-cell leukemia virus type 1 (HTLV-1) causes adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) in a small percentage of infected individuals. ATL is often associated with general immune suppression and an impaired HTLV-1-specific T-cell response, an important host defense system. We previously found that a small fraction of asymptomatic HTLV-1-carriers (AC) already showed impaired T-cell responses against the major target antigen, Tax. However, it is unclear whether the impaired HTLV-1 Tax-specific T-cell response in these individuals is an HTLV-1-specific phenomenon, or merely reflects general immune suppression. In this study, in order to characterize the impaired HTLV-1-specific T-cell response, we investigated the function of Tax-specific CD8⁺ T-cells in various clinical status of HTLV-1 infection.

Results

By using tetramers consisting of HLA-A*0201, -A*2402, or -A*1101, and corresponding Tax epitope peptides, we detected Tax-specific CD8⁺ T-cells in the peripheral blood from 87.0% of ACs (n = 20/23) and 100% of HAM/TSP patients (n = 18/18) tested. We also detected Tax-specific CD8⁺ T-cells in 38.1% of chronic type ATL (cATL) patients (n = 8/21), although its frequencies in peripheral blood CD8⁺ T cells were significantly lower than those of ACs or HAM/TSP patients. Tax-specific CD8⁺ T-cells detected in HAM/TSP patients proliferated well in culture and produced IFN-γ when stimulated with Tax peptides. However, such functions were severely impaired in the Tax-specific CD8⁺ T-cells detected in cATL patients. In ACs, the responses of Tax-specific CD8⁺ T-cells were retained in most cases. However, we found one AC sample whose Tax-specific CD8⁺ T-cells hardly produced IFN-γ, and failed to proliferate and express activation (CD69) and degranulation (CD107a) markers in response to Tax peptide. Importantly, the same AC sample contained cytomegalovirus (CMV) pp65-specific CD8⁺ T-cells that possessed functions upon CMV pp65 peptide stimulation. We further examined additional samples of two smoldering type ATL patients and found that they also showed dysfunctions of Tax-specific but not CMV-specific CD8⁺ T-cells.

Conclusions

These findings indicated that Tax-specific CD8⁺ T-cells were scarce and dysfunctional not only in ATL patients but also in a limited AC population, and that the dysfunction was selective for HTLV-1-specifc CD8⁺ T-cells in early stages.     

Altered neurochemical profile in the McGill‐R‐Thy1‐APP rat model of Alzheimer's disease: a longitudinal in vivo 1H MRS study

We investigated metabolite levels during the progression of pathology in McGill‐R‐Thy1‐APP rats, a transgenic animal model of Alzheimer's disease, and in healthy age‐matched controls. Rats were subjected to in vivo ¹H magnetic resonance spectroscopy (MRS) of the dorsal hippocampus at age 3, 9 and 12 months and of frontal cortex at 9 and 12 months. At 3 months, a stage in which only Aβ oligomers are present, lower glutamate, myo‐inositol and total choline content were apparent in McGill‐R‐Thy1‐APP rats. At age 9 months, lower levels of glutamate, GABA, N‐acetylaspartate and total choline and elevated myo‐inositol and taurine were found in dorsal hippocampus, whereas lower levels of glutamate, GABA, glutamine and N‐acetylaspartate were found in frontal cortex. At age 12 months, only the taurine level was significantly different in dorsal hippocampus, whereas taurine, myo‐inositol, N‐acetylaspartate and total creatine levels were significantly higher in frontal cortex. McGill‐R‐Thy1‐APP rats did not show the same changes in metabolite levels with age as displayed in the controls, and overall, prominent and complex metabolite differences were evident in this transgenic rat model of Alzheimer's disease. The findings also demonstrate that in vivo ¹H MRS is a powerful tool to investigate disease‐related metabolite changes in the brain.     

Family-wide chemical profiling and structural analysis of PARP and tankyrase inhibitors

Inhibitors of poly-ADP-ribose polymerase (PARP) family proteins are currently in clinical trials as cancer therapeutics, yet the specificity of many of these compounds is unknown. Here we evaluated a series of 185 small-molecule inhibitors, including research reagents and compounds being tested clinically, for the ability to bind to the catalytic domains of 13 of the 17 human PARP family members including the tankyrases, TNKS1 and TNKS2. Many of the best-known inhibitors, including TIQ-A, 6(5H)-phenanthridinone, olaparib, ABT-888 and rucaparib, bound to several PARP family members, suggesting that these molecules lack specificity and have promiscuous inhibitory activity. We also determined X-ray crystal structures for five TNKS2 ligand complexes and four PARP14 ligand complexes. In addition to showing that the majority of PARP inhibitors bind multiple targets, these results provide insight into the design of new inhibitors.     

Membrane-based actuation for high-speed single molecule force spectroscopy studies using AFM

Atomic force microscopy (AFM)-based dynamic force spectroscopy of single molecular interactions involves characterizing unbinding/unfolding force distributions over a range of pulling speeds. Owing to their size and stiffness, AFM cantilevers are adversely affected by hydrodynamic forces, especially at pulling speeds >10 μm/s, when the viscous drag becomes comparable to the unbinding/unfolding forces. To circumvent these adverse effects, we have fabricated polymer-based membranes capable of actuating commercial AFM cantilevers at speeds ≥100 μm/s with minimal viscous drag effects. We have used FLUENT^®, a computational fluid dynamics (CFD) software, to simulate high-speed pulling and fast actuation of AFM cantilevers and membranes in different experimental configurations. The simulation results support the experimental findings on a variety of commercial AFM cantilevers and predict significant reduction in drag forces when membrane actuators are used. Unbinding force experiments involving human antibodies using these membranes demonstrate that it is possible to achieve bond loading rates ≥10⁶ pN/s, an order of magnitude greater than that reported with commercial AFM cantilevers and systems.