High-performance affinity beads for identifying drug receptors

We have developed a method using novel latex beads for rapid identification of drug receptors using affinity purification. Composed of a glycidylmethacrylate (GMA) and styrene copolymer core with a GMA polymer surface, the beads minimize nonspecific protein binding and maximize purification efficiency. We demonstrated their performance by efficiently purifying FK506-binding protein using FK506-conjugated beads, and found that the amount of material needed was significantly reduced compared with previous methods. Using the latex beads, we identified a redox-related factor, Ref-1, as a target protein of an anti-NF-kappaB drug, E3330, demonstrating the existence of a new class of receptors of anti-NF-kappaB drugs. Our results suggest that the latex beads could provide a tool for the identification and analysis of drug receptors and should therefore be useful in drug development.     

The Effector Domain of MARCKS Is a Nuclear Localization Signal that Regulates Cellular PIP2 Levels and Nuclear PIP2 Localization

Translocation to the nucleus of diacylglycerol kinase (DGK)– ζ is dependent on a sequence homologous to the effector domain of Myristoylated Alanine Rich C-Kinase Substrate (MARCKS). These data would suggest that MARCKS could also localize to the nucleus. A single report demonstrated immunofluorescence staining of MARCKS in the nucleus; however, further experimental evidence confirming the specific domain responsible for this localization has not been reported. Here, we report that MARCKS is present in the nucleus in GBM cell lines. We then over-expressed wild-type MARCKS (WT) and MARCKS with the effector domain deleted (ΔED), both tagged with V5-epitope in a GBM cell line with low endogenous MARCKS expression (U87). We found that MARCKS-WT localized to the nucleus, while the MARCKS construct without the effector domain remained in the cytoplasm. We also found that over-expression of MARCKS-WT resulted in a significant increase in total cellular phosphatidyl-inositol (4,5) bisphosphate (PIP₂) levels, consistent with prior evidence that MARCKS can regulate PIP₂ levels. We also found increased staining for PIP₂ in the nucleus with MARCKS-WT over-expression compared to MARCKS ΔED by immunofluorescence. Interestingly, we observed MARCKS and PIP₂ co-localization in the nucleus. Lastly, we found changes in gene expression when MARCKS was not present in the nucleus (MARCKS ΔED). These data indicate that the MARCKS effector domain can function as a nuclear localization signal and that this sequence is critical for the ability of MARCKS to regulate PIP₂ levels, nuclear localization, and gene expression. These data suggests a novel role for MARCKS in regulating nuclear functions such as gene expression.     

Amino acid substitutions at protein–protein interfaces that modulate the oligomeric state

Despite similarities in their sequence and structure, there are a number of homologous proteins that adopt various oligomeric states. Comparisons of these homologous protein pairs, in terms of residue substitutions at the protein–protein interfaces, have provided fundamental characteristics that describe how proteins interact with each other. We have prepared a dataset composed of pairs of related proteins with different homo‐oligomeric states. Using the protein complexes, the interface residues were identified, and using structural alignments, the shadow‐interface residues have been defined as the surface residues that align with the interface residues. Subsequently, we investigated residue substitutions between the interfaces and the shadow interfaces. Based on the degree of the contributions to the interactions, the aligned sites of the interfaces and shadow interfaces were divided into primary and secondary sites; the primary sites are the focus of this work. The primary sites were further classified into two groups (i.e. exposed and buried) based on the degree to which the residue is buried within the shadow interfaces. Using these classifications, two simple mechanisms that mediate the oligomeric states were identified. In the primary‐exposed sites, the residues on the shadow interfaces are replaced by more hydrophobic or aromatic residues, which are physicochemically favored at protein–protein interfaces. In the primary‐buried sites, the residues on the shadow interfaces are replaced by larger residues that protrude into other proteins. These simple rules are satisfied in 23 out of 25 Structural Classification of Proteins (SCOP) families with a different‐oligomeric‐state pair, and thus represent a basic strategy for modulating protein associations and dissociations. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Identification of a novel GPR119 agonist, AS1269574, with in vitro and in vivo glucose-stimulated insulin secretion

The G protein-coupled receptor 119 (GPR119) is highly expressed in pancreatic β-cells. On activation, this receptor enhances the effect of glucose-stimulated insulin secretion (GSIS) via the elevation of intracellular cAMP concentrations. Although GPR119 agonists represent promising oral antidiabetic agents for the treatment of type 2 diabetes therapy, they suffer from the inability to adequately directly preserve β-cell function. To identify a new structural class of small-molecule GPR119 agonists with both GSIS and the potential to preserve β-cell function, we screened a library of synthetic compounds and identified a candidate molecule, AS1269574, with a 2,4,6-tri-substituted pyrimidine core. Here, we examined the preliminary in vitro and in vivo effects of AS1269574 on insulin secretion and glucose tolerance. AS1269574 had an EC₅₀ value of 2.5 μM in HEK293 cells transiently expressing human GPR119 and enhanced insulin secretion in the mouse pancreatic β-cell line MIN-6 only under high-glucose (16.8 mM) conditions. This contrasted with the action of the sulfonylurea glibenclamide, which also induced insulin secretion under low-glucose conditions (2.8 mM). In in vivo studies, a single administration of AS1269574 to normal mice reduced blood glucose levels after oral glucose loading based on the observed insulin secretion profiles. Significantly, AS1269574 did not affect fed and fasting plasma glucose levels in normal mice. Taken together, these results suggest that AS1269574 represents a novel structural class of small molecule, orally administrable GPR119 agonists with GSIS and promising potential for the treatment of type 2 diabetes.     

Charade of the SR K-Channel: Two Ion-Channels, TRIC-A and TRIC-B, Masquerade as a Single K-Channel

The presence of a sarcoplasmic reticulum (SR) K⁺-selective ion-channel has been known for >30 years yet the molecular identity of this channel has remained a mystery. Recently, an SR trimeric intracellular cation channel (TRIC-A) was identified but it did not exhibit all expected characteristics of the SR K⁺-channel. We show that a related SR protein, TRIC-B, also behaves as a cation-selective ion-channel. Comparison of the single-channel properties of purified TRIC-A and TRIC-B in symmetrical 210 mM K⁺ solutions, show that TRIC-B has a single-channel conductance of 138 pS with subconductance levels of 59 and 35 pS, whereas TRIC-A exhibits full- and subconductance open states of 192 and 129 pS respectively. We suggest that the K⁺-current fluctuations observed after incorporating cardiac or skeletal SR into bilayers, can be explained by the gating of both TRIC-A and TRIC-B channels suggesting that the SR K⁺-channel is not a single, distinct entity. Importantly, TRIC-A is regulated strongly by trans-membrane voltage whereas TRIC-B is activated primarily by micromolar cytosolic Ca²⁺ and inhibited by luminal Ca²⁺. Thus, TRIC-A and TRIC-B channels are regulated by different mechanisms, thereby providing maximum flexibility and scope for facilitating monovalent cation flux across the SR membrane.     

Removal of chromium by some multipurpose tree seedlings of Indian thar desert

An experiment was conducted to study the potential of chromium (Cr) phytoaccumulatory capabilities of four tree species viz., Anogeissus latifolia, Terminalia arjuna, Tecomella undulata, and Salvadora persica Possibility of enhancement of Cr uptake by citric acid and vesicular arbuscular mycorrhizal fungi (VAM) amendments were also tried. Cr is a major pollutant of the environment. Chromium can exist in oxidation states from III to VI, but the most stable and common forms of Cr are trivalent and hexavalent species. Cr(VI) was more toxic to the tree growth in terms of collar diameter (CD) increment in all the tree species than Cr(lll). Roots accumulated more Cr than shoots in all the tree species. There was more than 10 fold increase in root Cr content in comparison with shoot Cr content in all the trees at all the concentration of Cr and all sources of Cr. Citric acid significantly increased the Cr content in the tissues of roots in all the species under both speciation of Cr. The highest increase in Cr content brought by 20 mM citric acid addition was in A. latifolia Results suggest that Anogeissus latifolia is a potential Cr accumulator with citric acid as soil amendment.     

Polygalacturonase mRNA of Tomato: Size and Content in Ripe Fruits

In ripe tomato fruits, polygalacturonase (PG) mRNA comprisedabout 1% of the translatable RNAs in the poly(A)(+)RNA fraction.Sucrose density gradient centrifugation showed that this PGmRNA is similar in size to 18S rRNA, which suggests the presenceof a non-coding region. (Received June 19, 1984; Accepted October 23, 1984)     

Solid cancer incidence in atomic bomb survivors: 1958-1998

This is the second general report on radiation effects on the incidence of solid cancers (cancers other than malignancies of the blood or blood-forming organs) among members of the Life Span Study (LSS) cohort of Hiroshima and Nagasaki atomic bomb survivors. The analyses were based on 17,448 first primary cancers (including non-melanoma skin cancer) diagnosed from 1958 through 1998 among 105,427 cohort members with individual dose estimates who were alive and not known to have had cancer prior to 1958. Radiation-associated relative risks and excess rates were considered for all solid cancers as a group, for 19 specific cancer sites or groups of sites, and for five histology groups. Poisson regression methods were used to investigate the magnitude of the radiation-associated risks, the shape of the dose response, how these risks vary with gender, age at exposure, and attained age, and the evidence for inter-site variation in the levels and patterns of the excess risk. For all solid cancers as a group, it was estimated that about 850 (about 11%) of the cases among cohort members with colon doses in excess of 0.005 Gy were associated with atomic bomb radiation exposure. The data were consistent with a linear dose response over the 0- to 2-Gy range, while there was some flattening of the dose response at higher doses. Furthermore, there is a statistically significant dose response when analyses were limited to cohort members with doses of 0.15 Gy or less. The excess risks for all solid cancers as a group and many individual sites exhibit significant variation with gender, attained age, and age at exposure. It was estimated that, at age 70 after exposure at age 30, solid cancer rates increase by about 35% per Gy (90% CI 28%; 43%) for men and 58% per Gy (43%; 69%) for women. For all solid cancers as a group, the excess relative risk (ERR per Gy) decreases by about 17% per decade increase in age at exposure (90% CI 7%; 25%) after allowing for attained-age effects, while the ERR decreased in proportion to attained age to the power 1.65 (90% CI 2.1; 1.2) after allowing for age at exposure. Despite the decline in the ERR with attained age, excess absolute rates appeared to increase throughout the study period, providing further evidence that radiation-associated increases in cancer rates persist throughout life regardless of age at exposure. For all solid cancers as a group, women had somewhat higher excess absolute rates than men (F:M ratio 1.4; 90% CI 1.1; 1.8), but this difference disappears when the analysis was restricted to non-gender-specific cancers. Significant radiation-associated increases in risk were seen for most sites, including oral cavity, esophagus, stomach, colon, liver, lung, non-melanoma skin, breast, ovary, bladder, nervous system and thyroid. Although there was no indication of a statistically significant dose response for cancers of the pancreas, prostate and kidney, the excess relative risks for these sites were also consistent with that for all solid cancers as a group. Dose-response estimates for cancers of the rectum, gallbladder and uterus were not statistically significant, and there were suggestions that the risks for these sites may be lower than those for all solid cancers combined. However, there was emerging evidence from the present data that exposure as a child may increase risks of cancer of the body of the uterus. Elevated risks were seen for all of the five broadly classified histological groups considered, including squamous cell carcinoma, adenocarcinoma, other epithelial cancers, sarcomas and other non-epithelial cancers. Although the data were limited, there was a significant radiation-associated increase in the risk of cancer occurring in adolescence and young adulthood. In view of the persisting increase in solid cancer risks, the LSS should continue to provide important new information on radiation exposure and solid cancer risks for at least another 15 to 20 years.