Development of an HPLC Method with an ODS Column to Determine Low Levels of Aspartame Diastereomers in Aspartame

α-L-Aspartyl-D-phenylalanine methyl ester (L, D-APM) and α-D-aspartyl-L-phenylalanine methyl ester (D, L-APM) are diastereomers of aspartame (N-L-α-Aspartyl-L-phenylalanine-1-methyl ester, L, L-APM). The Joint FAO/WHO Expert Committee on Food Additives has set 0.04 wt% as the maximum permitted level of the sum of L, D-APM and D, L-APM in commercially available L, L-APM. In this study, we developed and validated a simple high-performance liquid chromatography (HPLC) method using an ODS column to determine L, D-APM and D, L-APM in L, L-APM. The limits of detection and quantification, respectively, of L, D-APM and D, L-APM were found to be 0.0012 wt% and 0.004 wt%. This method gave excellent accuracy, repeatability, and reproducibility in a recovery test performed on five different days. Moreover, the method was successfully applied to the determination of these diastereomers in commercial L, L-APM samples. Thus, the developed method is a simple, useful, and practical tool for determining L, D-APM and D, L-APM levels in L, L-APM.     

Characterization of conserved arginine residues on Cdt1 that affect licensing activity and interaction with Geminin or Mcm complex

All organisms ensure once and only once replication during S phase through a process called replication licensing. Cdt1 is a key component and crucial loading factor of Mcm complex, which is a central component for the eukaryotic replicative helicase. In higher eukaryotes, timely inhibition of Cdt1 by Geminin is essential to prevent rereplication. Here, we address the mechanism of DNA licensing using purified Cdt1, Mcm and Geminin proteins in combination with replication in Xenopus egg extracts. We mutagenized the 223th arginine of mouse Cdt1 (mCdt1) to cysteine or serine (R-S or R-C, respectively) and 342nd and 346th arginines constituting an arginine finger-like structure to alanine (RR-AA). The RR-AA mutant of Cdt1 could not only rescue the DNA replication activity in Cdt1-depleted extracts but also its specific activity for DNA replication and licensing was significantly increased compared to the wild-type protein. In contrast, the R223 mutants were partially defective in rescue of DNA replication and licensing. Biochemical analyses of these mutant Cdt1 proteins indicated that the RR-AA mutation disabled its functional interaction with Geminin, while R223 mutations resulted in ablation in interaction with the Mcm2～7 complex. Intriguingly, the R223 mutants are more susceptible to the phosphorylation-induced inactivation or chromatin dissociation. Our results show that conserved arginine residues play critical roles in interaction with Geminin and Mcm that are crucial for proper conformation of the complexes and its licensing activity.     

Intramolecular and intermolecular perturbation on electronic state of FAD free in solution and bound to flavoproteins: FTIR spectroscopic study by using the C = O stretching vibrations as probes

The intramolecular and intermolecular perturbation on the electronic state of FAD was investigated by FTIR spectroscopy by using the C=O stretching vibrations as probes in D(2)O solution. Natural and artificial FADs, i.e. 8-CN-, 8-Cl-, 8-H-, 8-OCH(3)-, and 8-NH(2)-FAD labelled by 2-(13)C, (18)O=C(2), or 4,10a-(13)C(2) were used for band assignments. The C(2)=O and C(4)=O stretching vibrations of oxidized FAD were shifted systematically by the substitution at the 8-position, i.e. the stronger the electron-donating ability (NH(2) > OCH(3) > CH(3) > H > Cl > CN) of the substituent, the lower the wavenumber region where both the C(2)=O and C(4)=O bands appear. In contrast, the C(4)=O band of anionic reduced FAD scarcely shifted. The 1,645-cm(-1) band containing C(2)=O stretching vibration shifted to 1,630 cm(-1) in the medium-chain acyl-CoA dehydrogenase (MCAD)-bound state, which can be explained by hydrogen bonds at C(2)=O of the flavin ring. The band was observed at 1,607 cm(-1) in the complex of MCAD with 3-thiaoctanoyl-CoA. The 23 cm(-1) shift was explained by the charge-transfer interaction between oxidized flavin and the anionic acyl-CoA. In the case of electron-transferring flavoprotein, two bands associated with the C(4)=O stretching vibration were obtained at 1,712 and 1,686 cm(-1), providing evidence for the multiple conformations of the protein.     

Crystal structure of the rac activator, Asef, reveals its autoinhibitory mechanism

The Rac-specific guanine nucleotide exchange factor (GEF) Asef is activated by binding to the tumor suppressor adenomatous polyposis coli mutant, which is found in sporadic and familial colorectal tumors. This activated Asef is involved in the migration of colorectal tumor cells. The GEFs for Rho family GTPases contain the Dbl homology (DH) domain and the pleckstrin homology (PH) domain. When Asef is in the resting state, the GEF activity of the DH-PH module is intramolecularly inhibited by an unidentified mechanism. Asef has a Src homology 3 (SH3) domain in addition to the DH-PH module. In the present study, the three-dimensional structure of Asef was solved in its autoinhibited state. The crystal structure revealed that the SH3 domain binds intramolecularly to the DH domain, thus blocking the Rac-binding site. Furthermore, the RT-loop and the C-terminal region of the SH3 domain interact with the DH domain in a manner completely different from those for the canonical binding to a polyproline-peptide motif. These results demonstrate that the blocking of the Rac-binding site by the SH3 domain is essential for Asef autoinhibition. This may be a common mechanism in other proteins that possess an SH3 domain adjacent to a DH-PH module.     

Identification and characterization of plasma kallikrein-kinin system inhibitors from salivary glands of the blood-sucking insect Triatoma infestans

Two plasma kallikrein-kinin system inhibitors in the salivary glands of the kissing bug Triatoma infestans, designated triafestin-1 and triafestin-2, have been identified and characterized. Reconstitution experiments showed that triafestin-1 and triafestin-2 inhibit the activation of the kallikrein-kinin system by inhibiting the reciprocal activation of factor XII and prekallikrein, and subsequent release of bradykinin. Binding analyses showed that triafestin-1 and triafestin-2 specifically interact with factor XII and high molecular weight kininogen in a Zn2+-dependent manner, suggesting that they specifically recognize Zn2+-induced conformational changes in factor XII and high molecular weight kininogen. Triafestin-1 and triafestin-2 also inhibit factor XII and high molecular weight kininogen binding to negatively charged surfaces. Furthermore, they interact with both the N-terminus of factor XII and domain D5 of high molecular weight kininogen, which are the binding domains for biological activating surfaces. These results suggest that triafestin-1 and triafestin-2 inhibit activation of the kallikrein-kinin system by interfering with the association of factor XII and high molecular weight kininogen with biological activating surfaces, resulting in the inhibition of bradykinin release in an animal host during insect blood-feeding.     

Effect of gap junction-mediated intercellular communication on TGF-β induced epithelial-to-mesenchymal transition

Epithelial-to-mesenchymal transition (EMT) is the process in which epithelial cells lose cell polarity and cell adhesion with surrounding cells to obtain migratory and invasive abilities. On the other hand, the expression of connexin is decreased or lacked in the many types of tumor cells. This study examined the effect of gap junctional intercellular communication (GJIC) on EMT induced by the transforming growth factor-β1 (TGF-β1). To investigate the effect of GJIC on EMT in U2OS cells, smooth muscle 22-α (sm22α) promoter-driven luciferase reporter gene was introduced into Cx43-expressing cells (U2OS-Luc Cx43) and into the control parental cell line (U2OS-Luc). TGF-β1 induced the expression of EMT markers and the sm22α promoter activity of U2OS-Luc cells. Sm22α promoter activity of U2OS cells was neither dependent on the expression of Cx43 nor on the establishment of GJIC among U2OS cells. Furthermore, we found that the homocellular communication among tumor cells did not affected the tumor cell growth and migration. However, we revealed that tumor cell density was an important factor for tumor cells to acquire metastatic phenotype. Interestingly, the co-culture of U2OS cells with osteoblasts revealed that sm22α promoter activity was inhibited only by the GJIC established between these two cell types. These results suggest that normal osteoblast cells negatively regulate the EMT of tumor cells, at least in part. Thus, Cx43-mediated GJIC may have anti-metastatic activity in tumor cells. Our findings provide a new insight into the role of GJIC in cancer progression and metastasis and identify potential therapeutic targets for the treatment of cancer.     

Effects of dietary protein contents and habitual endurance exercise on supplemental leucine oxidation in mice

The effects of dietary protein contents and regular exercise on the oxidation of supplemented leucine were examined. In the short-term study, male BALB/cCrSlc mice were fed diets containing 0, 10, 20, 35, and 60% protein: energy ratios for 1 week. In the long-term study, exercised and sedentary mice were fed diets containing 20, 35, and 60% protein ratios for 9 weeks. After the feeding periods, the mice were a bolus administered oral supplements of l-[1-¹³C] leucine. Expired gas was analyzed, and oxidized leucine was expressed as a relative ¹³CO₂/¹²CO₂ ratio. In the short-term study, the peak ¹³CO₂/¹²CO₂ ratio significantly increased with diet protein concentrations. Moreover, the long-term study also showed that the peak ¹³CO₂/¹²CO₂ ratio was significantly increased by high protein diets in both exercised and sedentary mice. Our results indicate that supplemental leucine oxidation is associated with consumption of a high-protein diet, irrespective of exercise status.

Abbreviations: AUC: area under the curve; EX: exercise; RQ: respiratory quotient; SED: sedentary; VO₂/W: oxygen uptake per body weight     

Mercuric chloride mediates a protein sulfhydryl modification-based pathway of signal transduction for activating Src kinase which is independent of the phosphorylation / dephosphorylation of a carboxyl terminal tyrosine

Little is known about the regulatory mechanism of c-Src kinase in cells except the suggested regulation through phosphorylation and dephosphorylation of its carboxyl terminal tyrosine residue (Y527). We here demonstrated that exposure of NIH3T3 cells to mercuric chloride (HgCl₂) induces both aggregation and activation of Src kinase protein through a redox-linked mechanism. The aggregation of Src proteins was suggested to be induced by the sulfhydryl groups-to-Hg²⁺ reaction-mediated polymerization of cell membrane proteins to which the Src proteins associate noncovalently. The possibility was ruled out that the aggregation occurred secondarily to the promotion of protein tyrosine phosphorylation. Further study revealed that the Src kinase was activated by HgCl₂ at least in part independent of the known Csk kinase-linked or Y527-phosphorylation/dephosphorylation-mediated control. Correspondingly, CNBr cleavage mapping of phosphopeptides for autophosphorylated c-Src protein demonstrated selective promotion of phosphorylation at Y416 in HgCl₂-treated cells without obvious change in the phosphorylation level at Y527. These results suggest a unique protein sulfhydryl modification-based pathway of signal transduction for activating Src kinase in NIH3T3 cells. © 1996 Wiley-Liss, Inc.