Identification of the cyclamate interaction site within the transmembrane domain of the human sweet taste receptor subunit T1R3

The artificial sweetener cyclamate tastes sweet to humans, but not to mice. When expressed in vitro, the human sweet receptor (a heterodimer of two taste receptor subunits: hT1R2 + hT1R3) responds to cyclamate, but the mouse receptor (mT1R2 + mT1R3) does not. Using mixed-species pairings of human and mouse sweet receptor subunits, we determined that responsiveness to cyclamate requires the human form of T1R3. Using chimeras, we determined that it is the transmembrane domain of hT1R3 that is required for the sweet receptor to respond to cyclamate. Using directed mutagenesis, we identified several amino acid residues within the transmembrane domain of T1R3 that determine differential responsiveness to cyclamate of the human versus mouse sweet receptors. Alanine-scanning mutagenesis of residues predicted to line a transmembrane domain binding pocket in hT1R3 identified six residues specifically involved in responsiveness to cyclamate. Using molecular modeling, we docked cyclamate within the transmembrane domain of T1R3. Our model predicts substantial overlap in the hT1R3 binding pockets for the agonist cyclamate and the inverse agonist lactisole. The transmembrane domain of T1R3 is likely to play a critical role in the interconversion of the sweet receptor from the ground state to the active state.     

Alkaline phosphatase vs luciferase as secreted reporter molecules in vivo

Secreted alkaline phosphatase (SEAP) and Metridia luciferase (MLuc) are useful reporter molecules in vitro, but little is understood about their usefulness in vivo. In this study, we investigated in vivo activity of recombinant SEAP and MLuc in blood and urine. When SEAP-transfected cells or recombinant SEAP were injected into rats, substantial increase in the level of serum SEAP was observed. In contrast, activity of SEAP was not detected in urine of rats injected with either the SEAP-transfected cells or recombinant SEAP. SEAP activity was also undetectable in urine of SEAP-injected Nagase analbuminemic rats in which glomerular permeability to macromolecules is enhanced. When MLuc-transfected cells were implanted into rats, activity of MLuc was undetectable not only in urine but also in serum. Even immediately after intravenous injection of recombinant MLuc, activity of MLuc was not detected in serum. Subsequent experiments revealed that, in contrast to SEAP, MLuc was rapidly inactivated either by rat serum, fetal bovine serum, or human serum. Albumin was identified as the molecule responsible for the inhibition of MLuc activity. These data elucidated advantages and limitations of secreted reporter molecules SEAP and MLuc under in vivo situations.     

Study on the distribution of the MSY2 polymorphism in 9 Chinese populations

The distribution of the MSY2 polymorphism in Chinese populations was analyzed by PCR. The results showed that the MSY2*4 allele, whose frequency in the total material was found to be 94.95%, was the common allele, while the distribution of the MSY2*3 allele was significantly different in the 416 tested males from the 9 populations under study. Based on the chi2-analysis, a distinct diversity was found in the non-group populations. Diversities were also discovered between southern and northern groups and among southern groups. On the contrary, no difference concerning the diversity was detected among the northern populations. In conclusion, MSY2 proved to be an important genetic marker with regard to the study of the genetic structure of Chinese populations, and further evidence is given concerning the migration direction between South and North.     

Lysophosphatidic acid inhibits anti-Fas-mediated apoptosis enhanced by actin depolymerization in epithelial ovarian cancer

Conflicting reports exist on the effect of actin depolymerization in anti-Fas-induced apoptosis. Lysophosphatidic acid (LPA) has been found to inhibit apoptosis in variable cell types. In this study, we evaluated LPA's protective effects on anti-Fas-induced apoptosis enhanced by actin depolymerization and possible mechanisms in epithelial ovarian cancer. OVCAR3 cells were pretreated with vehicle or LPA, then treated with Cytochalasin D (Cyto D), followed with anti-Fas mAb to induce apoptosis. Cells were stained with apoptotic markers and analyzed by flow cytometry. We report that LPA inhibited anti-Fas-induced apoptosis enhanced by actin depolymerization. Immunoprecipition of Fas death-inducing signaling complex (DISC) and Western blot suggested that the actin depolymerization accelerated caspase-8 activation, while LPA inhibited the association and activation of caspase-8 at the DISC. LPA inhibited caspase-3 and 7 activation induced by anti-Fas and/or Cyto D in cytosols. Phosphorylation of ERK and Bad112 by LPA may play a role in preventing caspase-3 activation through mitochondrial pathway induced by Cyto D. Our investigation found that LPA inhibited anti-Fas-induced apoptosis enhanced by actin depolymerization, and LPA may protect epithelial ovarian cancer from immune cell attack and cytoskeleton disrupting reagents induced apoptosis through multiple pathways.     

Charge substitution shows that repulsive electrostatic interactions impede the oligomerization of Alzheimer amyloid peptides

The strong pH dependence of A beta oligomerization could arise from favorable intermolecular charge-charge interactions between His and carboxylate groups, or, alternatively, by mutual electrostatic repulsion of peptide molecules. To test between these two possibilities, the pH dependence of the oligomerization of A beta and three charge substitution variants with Asp, Glu and His substituted by Ala is measured. All four peptides oligomerize, as detected by thioflavin T fluorescence, turbidity, and amyloid fibril formation; therefore, specific charge-charge interactions are nonessential for oligomerization. The strong negative correlation between net charge and oligomerization indicates that electrostatic repulsion between A beta monomers impedes their association.     

Low-frequency Fourier spectrum for predicting membrane protein types

Cell membranes are vitally important to living cells. Although the infrastructure of biological membrane is provided by the lipid bilayer, membrane proteins perform most of the specific functions. Knowledge of membrane protein types often provides crucial hints toward determining the function of an uncharacterized membrane protein. With the avalanche of new protein sequences generated in the post-genomic era, it is highly demanded to develop a high throughput tool in identifying the type of newly found membrane proteins according to their primary sequences, so as to timely annotate them for reference usage in both basic research and drug discovery. To realize this, the key is to establish a powerful identifier that can catch their characteristic sequence patterns for different membrane protein types. However, it is not easy because they are buried in a pile of long and complicated sequences. In this paper, based on the concept of the pseudo-amino acid composition [K.C. Chou, PROTEINS: Struct., Funct., Genet. 43 (2001) 246-255], the low-frequency Fourier spectrum analysis is introduced. The merits by doing so are that the sequence pattern information can be more effectively incorporated into a set of discrete components, and that all the existing prediction algorithms can be straightforwardly used on such a formulation for protein samples. High success rates were observed by the re-substitution test, jackknife test, and independent dataset test, indicating that the low-frequency Fourier spectrum approach may become a very useful tool for membrane protein type prediction. The novel approach also holds a high potential for predicting many other attributes of proteins.     

Fluorescence imaging of the activity of glucose oxidase using a hydrogen-peroxide-sensitive europium probe

A method for optical imaging of the activity of glucose oxidase (GOx) using a fluorescent europium(III) tetracycline probe for hydrogen peroxide is presented. A decay time in the microsecond range and the large Stokes shift of 210 nm of the probe facilitate intensity-based, time-resolved, and decay-time-based imaging of glucose oxidase. Four methods for imaging the activity of GOx were compared, and rapid lifetime determination imaging was found to be the best in giving a linear range from 0.32 to 2.7 m Unit/mL. The detection limit is 0.32 m Unit/mL (1.7 ng mL(-1)) which is similar to that of the time-resolved (gated) imaging using a microtiterplate reader. Fluorescent imaging of the activity of GOx is considered to be a useful tool for GOx-based immunoassays with potential for high-throughput screening, immobilization studies, and biosensor array technologies.     

The non-MHC quantitative trait locus Cia5 contains three major arthritis genes that differentially regulate disease severity, pannus formation, and joint damage in collagen- and pristane-induced arthritis

Cia5 is a locus on rat chromosome 10 which regulates the severity of collagen- and pristane-induced arthritis (CIA and PIA). To refine the region toward positional identification, Cia5 subcongenic strains were generated and studied in PIA and CIA. The protective effect of the telomeric locus Cia5a was confirmed in both models. A second arthritis severity locus (Cia5d) was identified within the most centromeric portion of Cia5. DA.F344(Cia5d) rats had a significantly lower median arthritis severity index in PIA, but not in CIA, compared with DA. On histologic analyses DA.F344(Cia5a) and DA.F344(Cia5d) congenics with PIA preserved a nearly normal joint architecture compared with DA, including significant reduction in synovial hyperplasia, pannus, angiogenesis, inflammatory infiltration, bone and cartilage erosions. Cia5 and Cia5a synovial levels of IL-1beta mRNA were reduced. Although both DA.F344(Cia5) and DA.F344(Cia5a) rats were protected in CIA, the arthritis scores of DA.F344(Cia5) were significantly higher than those of DA.F344(Cia5a), suggesting the existence of a third locus where F344-derived alleles centromeric from Cia5a contribute to increased arthritis severity. The existence of the third locus was further supported by higher levels of autoantibodies against rat type II collagen in DA.F344(Cia5) congenics compared with DA.F344(Cia5a). Our results determined that Cia5 contains three major arthritis severity regulatory loci regulating central events in the pathogenesis of arthritis, and differentially influencing CIA and PIA. These loci are syntenic to regions on human chromosomes 17q and 5q implicated in the susceptibility to rheumatoid arthritis, suggesting that the identification of these genes will be relevant to human disease.