The αvβ3 is generally expressed in low levels on the epithelial cells and mature endothelial cells, but it is highly expressed in many solid tumors. The αvβ3 levels correlate well with the potential for tumor metastasis and aggressiveness, which make it an important biological target for development of antiangiogenic drugs, and molecular imaging probes for early tumor diagnosis. Over the last decade, many radiolabeled cyclic RGD peptides have been evaluated as radiotracers for imaging tumors by SPECT or PET. Even though they are called “αvβ3-targeted” radiotracers, the radiolabeled cyclic RGD peptides are also able to bind αvβ5, α5β1, α6β4, α4β1, and αvβ6 integrins, which may help enhance their tumor uptake due to the “increased receptor population.” This article will use the multimeric cyclic RGD peptides as examples to illustrate basic principles for development of integrin-targeted radiotracers and focus on different approaches to maximize their tumor uptake and T/B ratios. It will also discuss important assays for pre-clinical evaluations of the integrin-targeted radiotracers, and their potential applications as molecular imaging tools for noninvasive monitoring of tumor metastasis and early detection of the tumor response to antiangiogenic therapy.The integrin family comprises 24 transmembrane receptors, each a heterodimeric combination of one of 18α and one of 8β subunits. Their main function is to integrate the cell adhesion and interaction with the extracellular microenvironment with the intracellular signaling and cytoskeletal rearrangement through transmitting signals across the cell membrane upon ligand binding. Integrin α The main objective of tumor imaging is to achieve the following goals: (1) to detect the presence of tumor at early stage, (2) to distinguish between benign and malignant tumors, (3) to follow the tumor growth and tumor response to a specific therapy , (4) to predict success or failure of a specific therapeutic regimen, and (5) to access the prognosis of a particular tumor.Figure 99mTc compounds due to optimal nuclear properties of 99mTc and its easy availability at low cost . Despite poor nuclear properties, 64Cu is a viable alternative to 18F for research programs that wish to incorporate high sensitivity and spatial resolution of PET, but cannot afford to maintain the expensive isotope production infrastructure , lack of specificity (αvβ3 vs. αIIBβ3), and rapid degradation by proteases in serum. Cyclization of RGD peptides via the linkers, such as S-S disulfide, thioether, and rigid aromatic rings, leads to the increased receptor binding affinity and selectivity ; SAA = 7-amino-l-glyero-l-galacto-2,6-anhydro-7-deoxyheptanamide) was the first one under clinical investigation for visualization of αvβ3 expression in cancer patients  have not yet been studied in details (Sheldrake and Patterson It must be noted that cyclic RGD peptides bind not only α2 (RGD2) was the first cyclic RGD dimer for development of diagnostic (99mTc) and therapeutic (90Y and 64Cu) radiotracers  multimeric cyclic RGD peptides have higher αvβ3 binding affinity and better tumor uptake than their monomeric analogs ]2, it is unlikely that they would bind to two adjacent αvβ3 sites simultaneously. However, the binding of one RGD motif to αvβ3 will increase the “local concentration” of second RGD motif in the vicinity of αvβ3 sites  E[c(RGDfK)]2 and E[c(RGDyK)]2 than their monomeric derivatives (Beer and Chen 4-E[c(RGDfK(PEG4))]2 , and 26 bonds G3-E[c(RGDfK(G3))]2 (3G-RGD2: G3 = Gly-Gly-Gly), which are long enough for them to achieve the bivalency. As a result, HYNIC-3P-RGD2 (IC50 = 60 ± 3 nmol/L) and HYNIC-3G-RGD2 (IC50 = 59 ± 3 nmol/L) have much higher αvβ3 binding affinity than HYNIC-P-RGD2 (P-RGD2 = PEG4-E[c(RGDfK)]2: (IC50 = 89 ± 7 nmol/L)) ]tes Fig. B. The co 90Y and 4Cu radioGD2 Fig. . Since tvβ3, αvβ5, α5β1, α6β4, α4β1, and αvβ6). Another approach  receptor. By targeting two different receptors, the radiotracer will have more opportunities to localize in the tumor due to the larger populations of two receptors than that of a single receptor. The so-called “bivalent heterodimers” -BBN(7–14) and c(RGDyK)-BBN(7–14) conjugates are “bivalent” for tumor targeting, and whether there is indeed a “synergetic effect” between the cyclic RGD and BBN(7–14) peptides. Another challenge associated with the “bifunctional heterodimer concept” is which binding unit actually contributes to the radiotracer tumor uptake.Two most important factors affecting the radiotracer tumor uptake are receptor binding affinity and receptor population. The receptor binding affinity is critically important for selective tumor localization and tumor uptake of radiolabeled cyclic RGD peptides  or RGD2) as the blocking agent. This experiment is often performed by biodistribution or imaging (PET or SPECT). The blocking agent is pre- or co-injected with the radiotracer. Co-injection or pre-injection of excess blocking agents (such as RGD2) will result in partial or complete blockage of the radiotracer tumor uptake . The normal organ uptake is consistent with the β3 and CD31 staining data for the liver, kidneys, and lungs from the tumor-bearing athymic nude mice.Blocking experiment Fig. A has beeake Fig. B. It is 125I-echistatin as the integrin-specific radioligand  was >20× lower than that of DOTA-3P-RGD2 (IC50 = 29 ± 4 nmol/L). Similar results were also seen with FITC-3P-RGK2 (IC50 = 589 ± 73 nmol/L) and FITC-3P-RGD2 (IC50 = 32 ± 7 nmol/L). Because of the low αvβ3 affinity of DOTA-3P-RGK2  had significantly lower (p < 0.01) uptake than 111In(DOTA-3P-RGD2) in the xenografted breast tumors and the αvβ3-positive normal organs, such as eyes, intestine, liver, lungs, and spleen  the in vitro binding assay using een Fig. . These rvβ3 level that will contribute the tumor uptake of a αvβ3-targeted radiotracer. The capability to visualize the αvβ3 expression provides new opportunities to characterize the tumor angiogenesis noninvasively, to select appropriate patients for antiangiogenic treatment, and to monitor the tumor response to antiangiogenic drugs. However, there were only a few reports on the correlation between the αvβ3 expression levels and radiotracer tumor uptake . IHC staining was performed to determine the αvβ3 and CD31 (a biomarker for tumor vasculature) expression levels in xenografted U87MG, MDA-MB-435, A549, HT29, and PC-3 tumor tissues   and T/M ratios of 99mTc-3P-RGD2 in the xenografted U87MG model, while no significant changes in tumor uptake of 99mTc-3P-RGD2 were seen in the PC-3 model after linifanib treatment  with low levels of αvβ3 and CD31 , antiangiogenic therapy would not be effective regardless the amount of antiangiogenic drug administered into the patient.D31 Fig. . Thus, 999mTc-3P-RGD2 SPECT/CT has been used as a noninvasive imaging tool to monitor the tumor growth and progression of breast cancer lung metastasis  with breast cancer lung metastasis. As expected, the SPECT/CT images showed no detectable metastatic breast tumor lesions in the lungs at week 4  uptake values of 99mTc-3P-RGD2 in the lungs. Even though the lung uptake of 99mTc-3P-RGD2 (0.41 ± 0.05 %ID) at week 4 seemed to be higher than that in the control animals (0.36 ± 0.06 %ID), this difference was not significant (p > 0.05) within the experimental errors. At week 6, the tumor burden in the lungs became significant. The lung uptake of 99mTc-3P-RGD2 was much higher  than that in the control group. By week 8, the uptake of 99mTc-3P-RGD2 in the lungs was increase to 1.40 ± 0.42 %ID. In all cases, the lung size remained relatively unchanged (1.21–1.32 cm3) during the 8-week study period.k 4 Fig. : top. Byk 4 Fig. . Figure 99mTc-3P-RGD2 in animal models guaranteed its further clinical application. In a first-in-human study, 99mTc-3P-RGD2 was investigated for its capability to noninvasively differentiate solitary pulmonary nodules (SPNs) . The sensitivity, specificity, and accuracy of 99mTc-3P-RGD2 SMM were 89.3%, 90.9%, and 89.7%, respectively, with a T/NT cut-off value of 2.40. The mean T/NT ratio of 99mTc-MIBI in malignant lesions was also significantly higher than that in benign lesions . The sensitivity, specificity, and accuracy of 99mTc-MIBI SMM were 87.5%, 72.7%, and 82.1%, respectively, with a T/NT cut-off value of 1.45. According to the ROC analysis, the area under the curve for 99mTc-3P-RGD2 SMM (area = 0.851) was higher than that for 99mTc-MIBI SMM (area = 0.781), but the statistical difference was not significant.The excellent in vivo tumor-targeting efficacy of 18F-labeled RGD compounds suffer from multistep and time-consuming synthetic procedures, which will limit their clinic availability. To overcome this shortcoming, the Al(NOTA) chelate has been used for 18F-labeling of P-RGD2  (denoted as 18F-Alfatide) was prepared in relatively high yield (42.1 ± 0.02) with more than 95% radiochemical purity. The whole radiosynthesis including post-labeling chromatographic purification was accomplished within 20 min. Nine patients with a primary diagnosis of lung cancer were examined by both static and dynamic PET imaging with 18F-alfatide, and one tuberculosis patient was investigated using both 18F-alfatide and 18F-FDG imaging. It was found that 18F-alfatide PET identified all tumors, with mean standardized uptake values of 2.90 ± 0.10. Tumor-to-muscle and tumor-to-blood ratios were 5.87 ± 2.02 and 2.71 ± 0.92, respectively. It was concluded that PET scanning with 18F-alfatide allows specific imaging of avb3 expression with good contrast in lung cancer patients.18F, 99mTc, 111In, 64Cu, and 68Ga) cyclic RGD peptides to target multiple integrins  will help to improve their tumor uptake due to the “increased receptor population.” In order to achieve bivalency, the distance between two cyclic RGD motifs must be long enough so that they will be able to bind the two adjacent αvβ3 sites simultaneously. Multimerization increases the uptake of radiolabeled multimeric cyclic RGD peptides in both the tumor and normal organs, and also their tumor retention times. Among the radiotracers evaluated in tumor-bearing models, the radiolabeled cyclic RGD dimers  show the most promising results with respect to their tumor uptake and T/B ratios. 99mTc-3P-RGD2, 18F-Alfatide, and 18F-Alfatide II are currently under clinical investigation for tumor imaging by SPECT or PET. 99mTc-3P-RGD2 offers significant advantages over both 18F-Alfatide and 18F-Alfatide II because it could be routinely prepared in high yield and radiochemical purity (>95%) without post-labeling chromatographic purification and clinical availability of 99Mo-99mTc generators. However, SPECT has limitations in quantification of radiotracer uptake, the speed of dynamic imaging, spatial resolution, and tissue attenuation.Over the last several years, many multimeric cyclic RGD peptides have been used to increase the radiotracer tumor-targeting capability. The fact that radiolabeled (


N,N′,N′′,N′′′-tetraisopropyl-1-(1-butylamidinato)guanidinato-κ3N1,N2:N2) bis[(tetrahydrofuran)lithium] and [bis(tetrahydrofuran)lithium]-di-μ-chlorido-holmate(III).The first metal complexes comprising amidinoguanidinate ligands have been prepared and structurally characterized, namely bisbis[(tetrahydrofuran)lithium], [Li2(C18H37N4)2(C4H8O)2], (2), and [bis(tetrahydrofuran)lithium]-di-μ-chlorido-{holmate(III)}, [HoLiCl2(C4H8O)2(C17H31N2)(C30H53N4)], (3). The novel lithium amidinoguanidinate precursors Li[nBuC(=NR)(NR)C(NR)2] Ho[nBuC(NCy)2](μ-Cl)2Li(THF)2 (3) in 71% yield. An X-ray crystal structure determination of 3 showed that this complex contains both an amidinate ligand and the new amidinoguanidinate ligand.The first metal complexes comprising amidinoguanidinate ligands have been prepared and structurally characterized, namely bis[μ- N-chelating donor ligands such as the amidinates [RC(NR)2]− and the guanidinates [R2NC(NR)2]− have gained tremendous importance in various fields of organometallic and coordination chemistry during the past two decades. Both types of N-chelating ligands are often regarded as ‘steric cyclopentadienyl equivalents’ (NR)C(NR)2] (1: R = Cy (cyclohexyl), 2: R = iPr) and the holmium(III) ‘ate’ complex [nBu-C(=NCy)(NCy)C(NCy)2]Ho[nBuC(NCy)2](μ-Cl)2Li(THF)2 (3).Anionic N,N′-dicyclohexylcarbodiimide and nBuLi in a 2:1 molar ratio in THF afforded the first lithium amidinoguanidinate, Li[nBuC(=NCy)(NCy)C(NCy)2]·THF (1), in 60% yield. This reaction represents the first case of dimerization of a carbodiimide under formation of a novel amidinoguanidinate anion. The lithium-amidinoguanidinate salt 1 is partially soluble in THF, Et2O, and DME and slightly soluble even in toluene and n-pentane. The new sterically bulky amidinoguanidinate 1 has been fully characterized by spectroscopic methods and elemental analysis to confirm the product as shown in Fig. 1d6 (DMSO = dimethyl sulfoxide) was found to be the best solvent for measuring the NMR spectra of Li[nBuC(=NCy)(NCy)C(NCy)2]·THF. A mass spectrum of 1 showed only fragments for the monomeric compound. Interestingly, the reaction using N,N′-dicyclohexylcarbodiimide and n-butyllithium in THF according to Fig. 11) could be isolated. A similar reaction carried out with N,N′-diisopropylcarbodiimide produced the isopropyl-substituted amidinoguanidinate salt 2 in >70% yield, although NMR data indicated the presence of significant amounts of an impurity, presumably the ‘normal’ lithium amidinate Li[nBuC(NiPr)2], which could not be separated by fractional crystallization from solvents like THF, DME or diethyl ether. However, occasionally a small amount of well-formed single-crystals of 2 were obtained directly from the reaction mixture which allowed a structural characterization of the new amidinoguanidinates through X-ray diffraction. Apparently the formation of the new amidinoguanidinate anions is critically influenced not only by the stoichiometric ratio of the starting materials, but also by the substituents at the N-atoms and the solvents employed. The solvent effect became apparent when reactions of N,N′-dicyclohexylcarbodiimide with 0.5 or 0.3 equiv. of n-butyllithium were carried out in Et2O solution. Using this solvent, the reactions produced a variable mixture of amidinoguanidinate and amidinate salts, Li[nBuC(=NCy)(NCy)C(NCy)2] and Li[nBuC(NCy)2], respectively, as illustrated in the reaction scheme (NCy)C(NCy)2] and Li[nBuC(NCy)2] with anhydrous HoCl3. In detail, treatment of N,N′-dicyclohexylcarbodiimide with 0.5 equiv. of nBuLi in Et2O followed by addition of anhydrous HoCl3 (NCy)C(NCy)2]Ho[nBuC(NCy)2](μ-Cl)2Li(THF)2 (3) in 71% yield. This compound is a mixed-ligand complex containing both the new amidinoguanidinato ligand and the normal amidinato ligand [nBuC(NCy)2]− in the coordination sphere of holmium. Compound 3 was fully characterized by its IR spectrum, elemental analysis and single-crystal X-ray diffraction. As a result of the highly paramagnetic nature of the Ho3+ ion, it was impossible to obtain interpretable NMR data for 3. Yellow, air- and moisture-sensitive, needle-like single-crystals of 3 were obtained by slowly cooling a saturated solution in n-pentane to 268 K.The presence of both types of anions in the reaction mixture obtained was also confirmed by the subsequent reaction of the N,N′-diorganocarbodiimides in the presences of sub-stoichiometric amounts of n-butyllithium. The cyclohexyl-substituted lithium-amidinoguanidinate salt Li[nBuC(=NCy)(NCy)C(NCy)2]·THF (1) is readily available as a pure solid in fairly good yield (60%). This compound could play an interesting role as a precursor for the synthesis of new transition metal and lanthanide amidinoguanidinate complexes. The first lanthanide complex comprising the new ligand system is the holmium ‘ate’ complex [nBuC(=NCy)(NCy)C(NCy)2]Ho[nBuC(NCy)2](μ-Cl)2Li(THF)2 (3).In summarizing the results reported here, we prepared the first metal complexes containing novel amidinoguanidinate ligands obtained by dimerization of 2 revealed the presence of ladder-type centrosymmetric dimers , which is the most characteristic structural motif of most previously characterized lithium amidinates and guanidinates . The Li—N distances within this ring are 2.0528 (17) and 2.1559 (17) Å and therefore in the expected range. The second N atom of the guanidinate unit (N1) is attached to only one Li atom with a shorter Li—N bond of 2.0177 (18) Å. Through this μ-κ3N,N′:N-coordination mode of the guanidinato moiety, a ‘ladder’ consisting of three four-membered rings is formed. By coordination of a solvent THF molecule, a typical distorted tetrahedral coordination of the Li atom is completed. The free N donor of the amidinate unit (N4) does not contribute to coordinative saturation of the Li atom. The bonds C1—N1 [1.3197 (12) Å] and C1—N2 [1.3396 (11) Å] are similar in length, indicating a common delocalization of the negative charge within the Li-coordinating N–C–N fragment. By contrast, the third C—N bond of the guanidinate unit C1—N3 is considerably longer at 1.4528 (11) Å and can therefore be interpreted as a pure single bond. The 1-butylamidinate fragment does not show any delocalization of the π-electron density, with one distinct double bond  and one single bond . The amidinate C3–C8–N3 fragment is twisted out of the guanidinate C1/N1/N2/N3 plane by approx. 75°, similar to that found earlier for this type of ligands (NCy)C(NCy)2]Ho[nBuC(NCy)2]Cl. The phenomenon of ‘ate’ complex formation via retention of alkali metal halides in the products is quite common in organolanthanide chemistry  ion is coordinated by two μ-bridging chloride ions, one chelating amidinoguanidinate ligand and one chelating amidinate ligand. The Ho atom is located in the C1N1N2N3 plane of the amidinoguanidinate ligand and, just like in the case of the lithium derivative 2, the amidinate N atom N4 does not contribute to metal coordination. The Ho—N distances are in a narrow range of 2.327 (3)–2.354 (3) Å that is in good agreement with the values observed in related lanthanide amidinate and guanidinate complexes 2] fragment is attached to the Ho atom in a formally chelating mode, leading to the formation of a regular kite-shaped Ho/Cl1/Li/Cl2 ring . The Li atom exhibits a typical tetrahedral coordination by the two μ-bridging Cl atoms and two THF ligands. Within the chelating NCN units of the amidinato and the amidinoguanidinato ligands, the C—N distances are nearly equal [1.324 (5)–1.336 (5) Å], indicating a typical π-electron delocalization within these units. The conformation of the amidinatoguanidinate ligand is very similar to that in compound 2 (angle between guanidinate and amidinate plane approx. 75°), and the localization of single and double bonds within the 1-butylamidinate backbone is identical with that in the lithium derivative [C—N 1.272 (5)–1.429 (5) Å].The holmium complex 2, the closest intermolecular contacts are between two isopropyl-CH3 groups  and between an isopropyl-CH3 and a butyl-CH3 group . The crystal structure of the holmium complex 3 comprises a close package of cyclohexyl groups, butyl groups and THF ligands with a minimal H2C⋯CH2 distance of 3.64 (4) Å, and one H2C⋯CH3 contact of at least 3.73 (6) Å (C6 and C21B of disordered cyclohexyl group).Due to an effective ‘packaging’ of the molecules by the sterically demanding alkyl substituents, both title compounds do not feature any specific intermolecular interactions. In the lithium derivative et al.  complexes with amidinate ligands, see: Richter  al. 2004, Edelman al. 2004, 2012 ▸ General Procedures: All reactions were carried out under an inert atmosphere of dry argon employing standard Schlenk and glovebox techniques. THF and n-pentane were distilled from sodium/benzophenone under nitrogen atmosphere prior to use. All glassware was oven-dried at 393 K for at least 24 h, assembled while hot, and cooled under high vacuum prior to use. Anhydrous holmium(III) chloride was prepared according to the literature method  and 13C NMR (100.6 MHz) spectra were recorded in DMSO-6d solution on a Bruker DPX 400 spectrometer at 298 K. Chemical shifts are referenced to TMS. IR spectra were recorded using KBr pellets on a Perkin Elmer FT–IR spectrometer system 2000 between 4000 cm−1 and 400 cm−1. Microanalyses  of compounds 1 and 3 were performed using a Leco CHNS 932 apparatus.Synthesis of Li[n2]·THF (1):BuC(=NCy)(NCy)C(NCy) A solution of N,N′-dicyclohexylcarbodiimide  in 100 ml of THF at 253 K was treated slowly with n-butyllithium . The reaction mixture was stirred for 10 min at 253 K, then warmed to room temperature and stirred overnight to give a white suspension in THF. The solvent was removed under vacuum affording 1 as white solid. Yield: 16.4 g, 60%. Elemental analysis for C34H61LiN4O (548.83 g mol−1): C, 74.41; H, 11.20; N, 10.21; found C, 74.82; H, 10.85; N, 10.50. 1H NMR 2SO, 298 K): δ (p.p.m.) 3.84 , 3.60 , 3.43 , 3.04–3.18 , 2.66 , 2.33 , 2.09 , 1.84 , 1.76 , 1.65 , 1.52 , 1.26 , 0.85 ; 13C NMR : δ (p.p.m.) 155.3 (NCN), 145.1 (NCN), 67.0 (THF), 55.4 , 54.2 , 49.3 , 35.7 , 35.1 , 34.8 , 34.5 , 30.7 , 29.5 , 25.8 (THF), 24.9 , 22.6 , 22.1 , 13.8 (CH3). MS : m/z (%) 125.2 (27) [Cy + C3H6]+, 153.2 (88) [2Cy − Me]2+, 183.3 (20) [2Cy + Me]2+, 207.3 (12) [C(NCy)2]+, 222.3 (62) [C(NCy)2 + Me]2+, 235.4 (100) [C(NCy)2 + C2H5]+, 264.4 (55) [nBu + C(NCy)2]+. IR (KBr): n (cm−1) 3449 (w), 3327 (w), 3225 (w), 2927 (vs), 2853 (s), 2666 (w), 2533 (w), 2354 (w), 2120 (w), 1959 (w), 1645 (m), 1578 (w), 1516 (m), 14450 (m), 1367 (w), 1339 (m), 1155 (w), 1128 (m), 1105 (w), 1053 (w), 1029 (w), 988 (w), 919 (w), 889 (w), 845 (w), 804 (w), 748 (w), 695 (w), 657 (w), 640 (w), 555 (w), 502 (w), 454 (w).Synthesis of Li[nBuC(=NiPr)(NiPr)C(Ni2]·THF (2):Pr) In a similar manner as for compound 1, N,N′-diisopropylcarbodiimide  was treated with n-butyllithium  in THF solution (80 ml). From this reaction 14.3 g of colorless 2 were isolated. X-ray quality single crystals (colorless rods) were occasionally obtained directly upon cooling of the reaction mixture to 278 K. However, NMR data showed that the bulk product was heavily contaminated with the lithium amidinate salt Li[nBuC(NiPr)2] (10–20%) which could not be separated by fractional crystallization.Synthesis of [n2]Ho[BuC(=NCy)(NCy)C(NCy)n2](BuC(NCy)μ2Li(THF)2 (3):-Cl) A solution of anhydrous HoCl3  in 50 ml THF was added to a stirred Et2O solution (80 ml) of an in situ-prepared mixture of Li[nBu-C(=NCy)(NCy)C(NCy)2] and Li[nBuC(NCy)2]  in 80 ml of Et2O and was treated slowly with n-butyllithium  at 253 K. The reaction mixture was stirred for 3 h at room temperature. The solvents were evaporated under vacuum, and the residue was extracted with 20 ml n-pentane. Concentration and cooling of the filtered solution to 278 K afforded 3 as yellow, air- and moisture-sensitive, needle-like crystals. Yield: 2.8 g, 71%. Elemental analysis for C55H100Cl2HoLiN6O2 (1120.22 g mol−1): C, 58.97; H, 9.00; N, 7.50; found C, 58.92; H, 8.98; N, 7.44%. IR (KBr): n (cm−1) 3321 (w), 3223 (w), 2929 (vs), 2857 (s), 2661 (w), 2525 (w), 2356 (w), 2118 (w), 1952 (w), 1577 (w), 1518 (m), 1367 (w), 1156 (w), 1129 (m), 1108 (w), 1085 (w), 1055 (w), 1045 (w), 983 (w), 922 (w), 892 (w), 865 (w), 820 (w), 715 (w), 657 (w), 643 (w), 553 (w), 505 (w), 456 (w). Meaningful NMR spectra could not be obtained due to the strong paramagnetism of the Ho3+ ion.Uiso(H) = 1.2 Ueq(C). C—H distances in CH3 groups were constrained to 0.98 Å, those in CH2 groups to 0.99 Å and those in CH groups to 1.00 Å. Methyl H atoms were allowed to rotate around the C—C vector but not to tip to best fit the experimental electron density (AFIX 137 in SHELXL). In the crystallographic dataset of compound 3, the intensities of reflections (3 are disordered. The aforementioned atoms were each split over two sites (site occupancy factors refined freely). Equivalent disordered THF and cyclohexyl moieties were restrained to have similar geometries (SAME restraint in SHELXLL), and Uij components of ADPs were restrained to be similar for atoms closer than 1.7 Å . Occupancy ratios refined to 0.760 (6) and 0.240 (6) for the cyclohexyl group (C19–C24), and to 0.663 (11) and 0.337 (11)  and to 0.823 (11) and 0.177 (11)  for the THF moieties.Crystal data, data collection and structure refinement details are summarized in Table 110.1107/S2056989016015322/zl2679sup1.cifCrystal structure: contains datablock(s) compound_2, compound_3. DOI: 10.1107/S2056989016015322/zl2679compound_2sup2.hklStructure factors: contains datablock(s) compound_2. DOI: 10.1107/S2056989016015322/zl2679compound_3sup3.hklStructure factors: contains datablock(s) compound_3. DOI: 1414136, 1414137CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


E)-3-(3-(carboxymethoxy)phenyl)acrylic acid (H2L) and 3-(pyridin-4-yl)pyrazole (pp) ligands, has been successfully synthesized under solvothermal conditions. In the crystal, helical chains formed by pp and L ligands connected to the Co metal, propagate parallel to the a axis.A two-dimensional polymeric structure based on ( 11H8O5)(C8H7N3)]n, which is based on (E)-3-[3-(carboxymethoxy)phenyl]acrylic acid (H2L) and 3-(pyridin-4-yl)pyrazole (pp) ligands, has been synthesized under solvothermal conditions. The dihedral angle between pyrazole and pyridine rings in the pp ligands is 23.1 (2)°. In the crystal, helical chains formed by pp and L ligands connected to the CoII atom propagate parallel to the c axis. CoII atoms of adjacent chains are bridged by the acrylic acid groups of L ligands into corrugated polymeric sheets in the ac plane.The title compound, [Co(C The CoII atom connects three L2− ligands and two pp ligands, and so can be described as a five-connected node. Thus, the topology of the structure could be given simply as a -connected network.In the structure, every ηL ligands form hydrogen bonds to the pp ligands, thereby enhancing the polymer stability -3-(3-carboxymethoxy)phenyl)acrylic acid and 1,3-di-pyridin-4-ylpropane ligands , recently reported by Wang et al. (C8H7N3)]Synthesis of [Co, H2L  mixed solvent. The pH value was adjusted to 7 by adding to a few drops of an aqueous NaOH solution (2.0 mol L−1). It was then sealed in a 25 mL stainless steel reactor and heated to 433 K for three days. The mixture was then cooled to room temperature at a rate of 5 K h−1, and red block-shaped crystals were obtained (yield: 62% based on Co). Analysis calculated (%) for C19H15CoN3O5 (424.27): C 53.81, H 3.62, N 9.85; found (%): C 53.79, H 3.56, N 9.90. IR data : 3432, 1649, 1501, 1407, 1274, 1206, 1180, 1086, 978, 844, 724, 603.(1): A mixture of CoClUiso(H) = 1.2Ueq(C) and C—H = 0.93 Å (aromatic and carbene) and 0.97 Å (methylene). Other hydrogen atoms were located in difference electron-density maps and refined freely.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S205698901601402X/pk2590sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S205698901601402X/pk2590Isup3.hklStructure factors: contains datablock(s) I. DOI: 1502210CCDC reference: crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The C=O and C—CN groups are trans to each other across their common C—C bond. In the crystal, one classical and two ‘weak’ hydrogen bonds combine with borderline N⋯N and S⋯S contacts to form layers parallel to C moiety is planar (r.m.s. deviation = 0.029 Å). The C=O and C—CN groups are trans to each other across their common C—C bond. In the crystal, one classical and two ‘weak’ hydrogen bonds combine with borderline N⋯N and S⋯S contacts to form layers parallel to (10-2). One ethyl group is disordered over two positions with relative occupancy 0.721/0.279 (7).In the molecule of the title compound, C S,S-acetals as potential starting materials for the preparation of novel classes of heterocycles has attracted much attention acetamide with carbon disulfide in the presence of sodium ethoxide followed by alkylation with ethyl iodide. The structure of (1) was originally based on its spectroscopic data and elemental analysis . In order to establish the structure of the compound unambiguously, the crystal structure was determined.The synthesis of ketene e.g. C9—C10 1.3781 (16) and C9—C11 1.4290 (16) Å]. The molecular backbone C1, N1, C8, C9, C10, S1, S2 is planar to within an r.m.s. deviation of 0.029 Å; O1 deviates by 0.063 (1) and C11 by 0.284 (1) Å from this plane. The aromatic ring subtends an angle of 53.30 (3)° with the same plane. The C=O and C—CN groups are trans to each other across the C8—C9 bond, with a torsion angle of 167.61 (11)°.The X-ray analysis confirms the exclusive presence of the form (1) in the solid state Fig. 1. Molecui connects the molecules across inversion centres; associated with this interaction, the N2 atoms of both molecules are forced into a close contact of 3.061 (2) Å. Two further contacts  Å is observed. All these secondary interactions combine to form a layer of molecules parallel to (10Hydrogen bonds are given in Table 10) Fig. 2.et al., 20162C=C(CN)C=O gave six hits . In all cases the C=O and C—CN groups are mutually trans, as in the title compound.A search of the Cambridge Database acetamide (1 mmol) was added to a stirred solution of potassium hydroxide (2 mmol) in DMF (10 ml). After stirring for 30 min at room temperature, carbon disulfide (1.5 mmol) was added. The solution was left for 12 h at room temperature and then ethyl iodide (2 mmol) was added dropwise. Stirring was continued for a further 6 h. The reaction mixture was poured onto ice–water and the solid product was filtered off, dried and crystallized from ethanol to give yellow crystals, m.p. 93°C (366 K), yield 40%.2-Cyano-−1; 1H NMR : δ 1.25 , 1.31 , 2.51 , 3.03 , 3.12 , 7.15–7.36 , 10.05 , Analysis calculated for C15H18ON2S2 (306.43): C, 58.82; H, 5.88, N, 9.15, S, 20.91%; Found: C, 58.70; H, 5.65, N, 9.00, S, 20.77%.IR (KBr), 3430 (NH), 2220 (CN), 1670 (C=O) cmCrystal data, data collection and structure refinement details are summarized in Table 2arom—H 0.95, Cmethylene—H 0.99 Å, with Uiso(H) = 1.5Ueq(C-methyl) and 1.2Ueq(C) for other H atoms.The NH hydrogen was refined freely. Methyl H atoms were refined as idealized rigid groups  allowed to rotate but not tip . Other hydrogen atoms were included using a riding model starting from calculated positions, with C10.1107/S2056989017005783/hg5488sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2056989017005783/hg5488Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989017005783/hg5488Isup3.cmlSupporting information file. DOI: 1544524CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


In the crystal, hydrogen bonds link adjacent molecules, forming a three-dimensional network of molecules stacked along the  7H13N3O3S [systematic name: (Z)-methyl 2-dimethylamino-N-(methylcarbamoyloxy)-2-oxoethanimidothioate], is an oxime carbamate acaride, insecticide and nematicide. The asymmetric unit comprises two independent molecules, A and B. The dihedral angles between the mean planes [r.m.s. deviations = 0.0017 (A) and 0.0016 Å (B)] of the acetamide and oxyimino groups are 88.80 (8)° for A and 87.05 (8)° for B. In the crystal, N/C—H⋯O hydrogen bonds link adjacent molecules, forming chains along the a axis. The chains are further linked by C—H⋯O hydrogen bonds, resulting in a three-dimensional network with alternating rows of A and B molecules in the bc plane stacked along the a-axis direction. The structure was refined as an inversion twin with a final BASF parameter of 0.16 (9).The title compound, C N,N-dimethyl-2-methylcarbamoyloximino-2-(dimethylsulfanyl)acetamide] is a carbamate compound used in a wide range of agricultural situations. It is systemic and active as an insecticide or a nematicide. It is used for the control of nematodes in vegetables, bananas, pineapple, peanuts, cotton, soya beans, tobacco, potatoes, sugar beet, and other crops. It is also used in India for controlling the growth of nematodes on vegetable crops  and 0.0016 Å (B)] of the acetamide and oxyimino groups are 88.80 (8) for A and 87.05 (8)° for B. All bond lengths and bond angles are normal and comparable to those observed in methomyl [systematic name: (E)-methyl N-(methylcarbamoyl)oxyethanimidothioate] which adopts similar crystal structure  = 0.88 Å, Uiso = 1.2Ueq(C) for N—H group, Uiso = 1.5Ueq(C) for methyl group, d(C—H) = 0.98 Å]. The crystal studied was an inversion twin with a 0.84 (9):0.16 (9) domain ratio.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989016018168/sj5514sup1.cifCrystal structure: contains datablock(s) I, New_Global_Publ_Block. DOI: 10.1107/S2056989016018168/sj5514Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989016018168/sj5514Isup3.cmlSupporting information file. DOI: 1516996CCDC reference: crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Three binary and one ternary charge-transfer complexes have been made using 1,3,5-trinitrobenzene, all of which show alternating donor and acceptor stacks, which have weak C—H⋯O hydrogen bonds perpendicular to the stacking axis. The final complex is a crystal engineering attempt to modify the packing of the stacks by inserting a third molecule into the structure; this third molecule features strong hydrogen bonds between the carboxylic acid group of the donor molecule and the pyridine acceptor molecule. viz. 1,3,5-trinitrobenzene–2-acetylnaphthalene (1/1), C6H3N3O6·C12H10O, (I), 1,3,5-trinitrobenzene–9-bromoanthracene (1/1), C14H9Br·C6H3N3O6, (II), 1,3,5-trinitrobenzene–methyl red (1/1), C15H15N3O2·C6H3N3O6, (III) (systematic name for methyl red: 2-{(E)-[4-(dimethylamino)phenyl]diazenyl}benzoic acid), and 1,3,5-trinitrobenzene–1-naphthoic acid–2-amino-5-nitropyridine (1/1/1), C6H3N3O6·C11H8O2·C5H5N3O2, (IV). All charge-transfer complexes show alternating donor and acceptor stacks, which have weak C—H⋯O hydrogen bonds perpendicular to the stacking axis. In addition, complex (IV) is a crystal engineering attempt to modify the packing of the stacks by inserting a third molecule into the structure. This third molecule is stabilized by strong hydrogen bonds between the carboxylic acid group of the donor molecule and the pyridine acceptor molecule.Three binary and one ternary charge-transfer complexes have been made using 1,3,5-trinitrobenzene,  Co-crystallization presents an opportunity to re-look at these problems for example, Guo l. 2013a have sho al. 2017 isolatedZ′ = 1 with all molecules in general positions. As a result of the strong polarizing effect of the nitro groups, TNB has an electron-poor π-system. On the other hand, the donor molecules (polycyclic aromatic hydrocarbons) have an electron-rich π-system. The packing of the unit cell of the complexes follows a donor (D) acceptor (A) π–π interaction, which is the major driving force in the formation of these complexes, as seen in Fig. 2D⋯A stacks can be quantified using Hirschfeld surface analysis as well as the resulting fingerprint plots using the programme CrystalExplorer17.5 . In the plane perpendicular to the stacking, the ac plane, there are C—H⋯O interactions between TNB and 2-acetylnaphthalene molecules (Table 3a), forming an et al., 1995Structure (I)ne Fig. 2a. In ths Table 3a, formiP21/c space group with both the TNB and 9-bromoanthracene in the asymmetric unit. The packing of the structure displays a clear separation of the donor (blue) and acceptor (green) layers . The alternating DA stacks show that the bromine atom of the 9-bromoanthracene packs in a head-to-head stacked fashion; however, the distance between the nearest Br atoms is very long [4.981 (1) Å], much longer than the sum of their van der Waals radii, and as a result Br⋯Br interactions are not involved in the molecular aggregation. In the plane perpendicular to the stacking, there are C—H⋯O interactions (Table 4b).Structure (II)rs Fig. 2b. The as Table 4 between es Fig. 4b.Pca 0.98 Å c), and an additional six-membered intramolecular S(6) hydrogen bond is found . The TNB and methyl red molecules are again joined by C—H⋯O hydrogen bonds (Table 5c).Structure (III)Å Table 2. Along tnd Fig. 1c. The Ts Table 5 to the nng Fig. 4c.PDA stacks separated by the added pyridine component . The TNB molecules are joined by DA stacks and the 2-amino-5-nitropyridine molecule was found, forming an d); interestingly there is no additional DA stacking with the 1-naphthoic and the pyridine components.Structure (IV)nt Fig. 2d. The Tg Table 6d; interIn summary, we have contributed to the field of CT complexes using TNB, presenting further evidence that TNB is an ideal acceptor and, when paired with a donor that has hydrogen-bonding functionality, can be used to make ternary complexes.et al., 2016A database survey in the Cambridge Structural Database  and used as received without further purification. The 1,3,5-trinitrobenzene charge-transfer complexes were prepared in a 10 mL ethanolic solution with a 1:1 or 1:1:1 stoichiometric ratio of the donor to the acceptor molecule (based on 0.469 mmol of TNB), which was then heated until total dissolution took place (approx. 4 h). The solution was then cooled very slowly to obtain crystals suitable for X-ray diffraction. Detailed masses are as follows: (I)3), and 0.95 Å (Ar—H)] and refined as riding with Uiso(H) = 1.2Ueq(Ar-C) or Uiso(H) = 1.5Ueq(methyl-C). The O and N–bound H atoms were located in the difference map and their coordinates and isotropic displacement parameters allowed to refine freely.Crystal data, data collection and structure refinement details are summarized in Table 710.1107/S2056989018000245/eb2004sup1.cifCrystal structure: contains datablock(s) I, II, III, IV, shelx. DOI: 10.1107/S2056989018000245/eb2004Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989018000245/eb2004IIsup3.hklStructure factors: contains datablock(s) II. DOI: 10.1107/S2056989018000245/eb2004IIIsup4.hklStructure factors: contains datablock(s) III. DOI: 10.1107/S2056989018000245/eb2004IVsup5.hklStructure factors: contains datablock(s) IV. DOI: 10.1107/S2056989018000245/eb2004sup6.pdfFingerprint plots. DOI: Click here for additional data file.10.1107/S2056989018000245/eb2004Isup7.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989018000245/eb2004IIsup8.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989018000245/eb2004IIIsup9.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989018000245/eb2004IVsup10.cmlSupporting information file. DOI: 1814637, 1814636, 1814635, 1814634CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Pseudomonas putida. The enzyme is composed of two opposing domains with Rossmann folds, thus constituting a B pattern-type glycosyltransferase (GT-B). While dTDP-β-l-rhamnose is located within a highly conserved pocket of the C-domain, EarP recognizes the KOW-like N-domain of EF-P. Based on our data, we propose a structural model for arginine glycosylation by EarP. As EarP is essential for pathogenicity in P. aeruginosa, our study provides the basis for targeted inhibitor design.Glycosylation is a universal strategy to posttranslationally modify proteins. The recently discovered arginine rhamnosylation activates the polyproline-specific bacterial translation elongation factor EF-P. EF-P is rhamnosylated on arginine 32 by the glycosyltransferase EarP. However, the enzymatic mechanism remains elusive. In the present study, we solved the crystal structure of EarP from  Pseudomonas aeruginosa infection.The structural and biochemical characterization of the EF-P-specific rhamnosyltransferase EarP not only provides the first molecular insights into arginine glycosylation but also lays the basis for targeted-inhibitor design against  Escherichia coli and Salmonella enterica—ß-lysinylate a conserved lysine, K34EF-P, by EpmA. This EF-P-specific ligase uses β-(R)-lysine as the substrate, which is generated by isomerization of α-(S)-lysine by employing the activity of the amino mutase EpmB  as donor substrate, resulting in α-rhamnosyl-arginine on the acceptor EF-P (E. coli (EPEC) was shown to inactivate human cell death domain-containing proteins by N-acetylglucosaminylation of arginine, with this being a major pathogenicity determinant during infection (earP abolishes the pathogenicity of P. aeruginosa (Translation elongation is a nonuniform process and directly depends on the amino acids (aa) to be incorporated into the growing polypeptide chain . Due to 5–8–ase EpmB –23. In cposition , 24, 25.tor EF-P , 27. Unltor EF-P , 29. To tor EF-P . In the nfection . Similarruginosa . AccordiPseudomonas putida KT2440 (EarPPpu) bound to its cognate nucleotide-sugar donor substrate TDP-Rha at a 2.3-Å resolution (PDB accession number 5NV8). Together with reporting the results of nuclear magnetic resonance (NMR) spectroscopy analyses and an in vitro/in vivo biochemical enzyme characterization, we lay the foundation for understanding arginine glycosylation.Here we present the X-ray crystal structure of EarP from 2 (–Pseudomonas putida (see N-acetylglucosamine (UDP-GlcNAc)-dependent glycosyltransferases MurG from E. coli (MurGEco) (Xanthomonas campestris (OGTXca) (Ppu adopts a clamp-like structure with two opposing Rossmann-like domains that are separated by an interdomain cleft (Despite low sequence conservation most nucleotide sugar dependent (Leloir-type) glycosyltransferases adopt one of two major folding patterns, GT-A or GT-B . However2 , and the2 –36 to geMurGEco)  and O-Gl(OGTXca)  as strucin cleft . With thin cleft .10.1128/mBio.01412-17.1FIG S1 Rha antibodies. One point five micrograms of unmodified EF-P (EF-P−) and 0.5 µg modified EF-P (EF-PRha) were subjected to SDS-PAGE and transferred to a nitrocellulose membrane by horizontal Western blotting. The membrane was cut, and the halves were used to detect EF-PPpu using 0.2 µg/ml anti-EF-P and EF-PRha using 0.25 µg/ml anti-ArgRha antibodies. (B) In vivo functionality analysis of EarPPpu single-amino-acid exchange variants. (Top) β-Galactosidase activity in E. coli MG1655 PcadBA::lacZ Δefp upon expression of EF-PPpu and EarPPpu and coexpression of EF-PPpu with EarPPpu and single-amino-acid substitution variants. Cells were incubated under cadBA-inducing conditions  at 30°C o/n . (Bottom) Rhamnosylation of EF-PPpu in MG1655 cells coexpressing EF-PPpu and its EarPPpu single-amino-acid exchange variants was assessed by Western blotting and detection of EF-PRha using 0.25 µg/ml anti-ArgRha. Download FIG S1, TIF file, 1.8 MB.(A) Specificity of anti-Arg30°C o/n . Data arCopyright © 2017 Krafczyk et al.2017Krafczyk et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.01412-17.2FIG S2 in vitro rhamnosylation of EF-PPpu by EarPPpu. Fixed amounts of EF-PPpu (2.5 µM), EarPPpu (0.1 µM), and TDP-Rha (500 µM) were incubated at 30°C in 100 mM NaPi, pH 7.6, for various amounts of time. Measurements were performed in technical duplicates. Standard errors are shown. A representative SDS-PAGE gel and Western blot used for generation of the time course curve are shown. Rhamnosylated EF-PPpu (EF-PRha) was detected using 0.25 µg/ml anti-ArgRha. Band intensities were quantified using ImageJ (Ppu single-amino-acid exchange variants. Fixed amounts of EF-PPpu (2.5 µM) and the respective EarPPpu single-amino-acid exchange variants (0.1 µM) were incubated with various concentrations of TDP-Rha at 30°C in 100 mM NaPi, pH 7.6. In the cases of the F191AEarP and Y1931AEarP variants, 0.5 µM concentrations were used. Suitable incubation times were determined by time course experiments prior to determination of kinetic parameters (data not shown). Representative SDS-PAGE gels and Western blots used for generation of the TDP-Rha saturation curves are depicted underneath the corresponding graphs. Rhamnosylated EF-PPpu was detected after Western blotting using 0.25 µg/ml anti-ArgRha. Band intensities were quantified using ImageJ  Time-dependent g ImageJ . (B) TDPg ImageJ . Km and Copyright © 2017 Krafczyk et al.2017Krafczyk et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.01412-17.3FIG S3 P. putida. (Top) Structural models of EarP from P. putida were generated using Phyre2 (orange), the I-TASSER server (white), and SWISS-MODEL (blue). Model coverage and scoring is shown underneath the respective model structures. (Bottom) Overlay of structural models from Phyre2 and I-TASSER (left), I-TASSER and SWISS-MODEL (middle), and SWISS-MODEL and Phyre2 (right). Root mean square deviations of atomic positions are shown underneath the respective structural overlays. All illustrations and overlays were generated using UCSF Chimera and the UCSF Chimera MatchMaker (σ is depicted as grey mesh. (D) SAXS analysis of free EarP and EarP bound to TDP-Rha. The two experimental small-angle X-ray scattering curves of free EarP (black) and EarP plus TDP-Rha (blue) do not exhibit any significant changes. Thus, it can be concluded that there are no major conformational changes upon ligand binding. The crystal structure and monomeric states of the protein are also validated in solution, as the back-calculated scattering densities (red line) fit well with the experimental data (χ2 = 1.96). The deviation at the low Q range is due to aggregation and/or interparticle interaction. (E) Secondary structure of EF-P from P. putida. The secondary structures of individual amino acids are indicated as a propensity to form either α-helix (red) or β-strand (green). The amino acids with a propensity to adopt a random coil or lacking information about secondary structure were assigned zero values in the plot. The propensity values were obtained from Cα, Cβ, and C′ secondary chemical shifts using CcpNmr Analysis (P. aeruginosa (PDB accession number 3OYY)  Fold recognition models of EarP from tchMaker . (B) B-fAnalysis . (F) Secer 3OYY) . Red ribCopyright © 2017 Krafczyk et al.2017Krafczyk et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Ppu at 2.3-Å resolution  and seven α-helices (α8 to α14) (2 versus 46 Å2)  . On the  Å2) see , model 2 Å2) see . In the  Å2) see . However Å2) see , model 210.1128/mBio.01412-17.8DATA SET S1 DATA SET S1, XLSX file, 0.02 MB.Strains, plasmids, and primers used in the study. Download Copyright © 2017 Krafczyk et al.2017Krafczyk et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.01412-17.9DATA SET S2 DATA SET S2, XLSX file, 0.01 MB.Crystallographic data collection and refinement statistics. Download Copyright © 2017 Krafczyk et al.2017Krafczyk et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.01412-17.4FIG S4 FIG S4, TIF file, 7.9 MB.(A) Stereo view of the EarP–TDP-Rha complex. Electron density is shown. (B and C) Electron densities for the N- and C-terminal domains, respectively. The N-terminal domain shows several disconnected blobs of electron density currently modeled with water, and some regions are entirely missing. Download Copyright © 2017 Krafczyk et al.2017Krafczyk et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Ppu at 43 kDa exceeds the sensitivity limitations of NMR, demanding deuteration in order to decrease cross-relaxation effects and to decrease the signal line width. Nonetheless, using transverse relaxation-optimized spectroscopy (TROSY)-based experiments, we were able to assign 62% of the EarPPpu backbone.Furthermore, the presence of the predicted strands and helices and thus the validity of the model and crystal structure could be confirmed by NMR secondary chemical shifts . A prereThe two domains are interconnected by a bipartite helix  comprising aa 156 to 176. This linker region together with an unstructured segment that positions α15 in the vicinity of the N terminus defines the floor of the cleft that separates the domains .carbohydrate-active enzymes (CAZy) database  STD NMR at a 1:100 ratio of protein to ligand shows several signals from the TDP but no signal from the sugar region of the TDP-Rha, thus confirming the crystal structure where the sugar is solvent exposed, and does not show extensive contacts with the protein. (B) STD NMR with a low excess of TDP-Rha (1:7 ratio of protein to ligand) clearly shows the signal for the methyl group of the thymidine ring, confirming the interaction between TDP-Rha and EarP under conditions used for SAXS data collection. (C) STD NMR experiments with E273 variants showing that the mutation of E273 to alanine, glutamine, or aspartate does not affect the binding of TDP-Rha to EarP. Download Copyright © 2017 Krafczyk et al.2017Krafczyk et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Ppu and EarPPpu bound to TDP-Rha has been performed  of free EarPerformed . The oveerformed ; howevererformed .Pseudomonadales, Aeromonadales, and Alteromonadales . Ph. Ph17). 10.1128/mBio.01412-17.10DATA SET S3 DATA SET S3, XLSX file, 0.1 MB.Collection of EarP homologues. Download Copyright © 2017 Krafczyk et al.2017Krafczyk et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the In order to identify conserved amino acids, we used Clustal Omega  and genePpu constructs with single-amino-acid substitutions of the individual residues forming the binding pocket and tested the activities of the EarPPpu variants both in vivo and in vitro  can restore β-galactosidase activity in an E. coli PcadBA::lacZ Δefp strain  and specifically detects arginine rhamnosylation   . This inanalysis  and D. Iectively , and D.Km makes sense physiologically and therefore analyzed the cellular TDP-Rha levels in P. putida, P. aeruginosa, and E. coli, which were 3.5 mM, 2.0 mM, and 4.0 mM, respectively , P. putida KT2440 (middle), and P. aeruginosa (bottom). Purified EF-PPpu (2.5 µM) and EarPPpu (0.1 µM) were incubated with increasing concentrations of TDP-Rha or lysates from ~107 and ~108 cells . Samples containing cell lysates were incubated in the presence and absence of EarPPpu. Rhamnosylated EF-P (EF-PRha) was detected using 0.25 µg/ml anti-ArgRha. For E. coli and P. aeruginosa, experiments were performed as biological triplicates. (B) Representative calibration curves calculated from relative Western blot band intensities in samples containing increasing TDP-Rha concentrations. Band intensities were quantified using ImageJ (8 lysed cells (triangles) were calculated from the slope of the calibration curve and are indicated by dashed lines. Band intensities from samples containing no EarPPpu were subtracted from those containing EarPPpu to correct for background signal. (C) Equations used to calculate average TDP-Rha concentrations in single cells. For calculation of average TDP-Rha concentrations in E. coli cells, an average cell volume of 3.9 fl was assumed  SDS-PAGE gels and Western blots for determination of the intracellular TDP-Rha concentration in g ImageJ . TDP-Rha assumed . For cal assumed . DownloaCopyright © 2017 Krafczyk et al.2017Krafczyk et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Km and kcat of EarPPpu substitution variants were determined and compared to those of the wild-type protein. Strikingly, all earP mutations affected enzymatic activity (Km increased up to 60-fold for the F252AEarP variant (Km = 3.4 mM). Conversely, the kcat decreased up to 3,500 times when we measured the kinetics of the F191AEarP and Y193AEarP variants.Next, the activity  and S2B.Ppu variants  were analyzed by NMR 1H-15N heteronuclear single quantum coherence (HSQC) experiments  in the protein N-domain (EarP variant by size exclusion chromatography (SEC) revealed an elution pattern with three distinct EarP peaks, indicating a certain degree of protein aggregation. However, the lowest molecular peak in the D274AEarP SEC profile is congruent with the one that we found when subjecting WTEarP to SEC. Accordingly, Km (TDP-Rha) and kcat values were determined from this protein fraction to be 206 µM and 0.74 min−1, respectively  and overlaid with a spectrum of wild-type protein (grey). Note that the mutant spectra were recorded with a small number of scans. Spectra of all variants except the D274A variant overlap well with the wild-type spectrum and show well-dispersed peaks (an indicator of a stable secondary structure), confirming that the single-amino-acid substitutions do not influence proper folding of EarP. In contrast, the D274A variant is in large part unfolded. Download FIG S7, TIF file, 3.1 MB.Assessing folding state of EarPCopyright © 2017 Krafczyk et al.2017Krafczyk et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Ppu and WTEarP as well as the above-mentioned nine substitution variants  are severely line broadened beyond detection. Therefore, chemical shift perturbation values cannot be determined for these and vicinal residues. This line broadening is an indication that they are bound by EarPPpu and thus have rotational correlation times expected for a complex of that size. Several residues located in the S1-like OB-domain are also slightly affected. However, this is not necessarily due to direct contacts with EarPPpu but might also be propagating effects. Therefore, we also investigated in vitro rhamnosylation of truncated EF-PPpu variants comprising either amino acids 1 to 128 or amino acids 1 to 65 . Th. Th1H-15F-P  exhibited strong perturbations not only in WTEarP but also in the D13AEarP and D17AEarP variants upon TDP-Rha binding, despite not forming direct ligand contacts -containing LB plates  specifically the arginines at positions 117 and 235 in the death domain-containing proteins FADD and TRADD, respectively  (3ZY6), where F357 is involved in π-stacking with the respective nucleobase  . Similarcleobase . Stackine amides , 54, 55.Eco (PDB accession number 1F0K)  strongly impairs protein function, all of which suggests that they have similar roles in product stabilization.In GT-B glycosyltransferases, positively charged amino acids are often involved in facilitating leaving group departure. This is achieved by neutralization of evolving negative charges on the phosphate moiety during the glycosyl transfer reaction, as described, e.g., for R261 of MurGer 1F0K) . NotablyEF-P. In agreement with this hypothesis, the EF-P β-lysine ligase EpmA, for example, recognizes EF-P via identity elements in a region located around the E. coli EF-P modification site K34  (P. putida) and 37°C (for E. coli), unless indicated otherwise. When required, media were solidified by using 1.5% (wt/vol) agar. If necessary, media were supplemented with 50 µg/ml chloramphenicol, 100 µg/ml kanamycin sulfate, and/or 100 µg/ml ampicillin sodium salt. For promoter induction from PBAD-containing plasmids  in liquid medium. For promoter induction from plasmids comprising the lac operator sequences, isopropyl β-d-1-thiogalactopyranoside (IPTG) (Sigma-Aldrich) was added to a final concentration of 1 mM.Strains and plasmids used in this study are listed in oth (LB) , 61 accooth (LB)  at 30°C plasmids , l-arabiearP gene by overlap extension PCR  overnight (o/n) and harvested by centrifugation. β-Galactosidase activities were determined as described in reference t test and stating a result as significantly different if P was <0.05.Cells expressing E. coli BTH101 cells were cotransformed with pUT18C-efpPpu and/or the respective pKT25 variants  and plated on LB screening medium containing 40 μg/ml 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside  and 0.5 mM IPTG as well as 50 µg/ml kanamycin sulfate and 100 µg/ml ampicillin sodium salt. Transformants containing pUT18-zip and pKT25-zip were used as positive controls. Transformants carrying pUT18C and pKT25 vector backbones were used as negative controls. Bacteria expressing interacting protein hybrids exhibit a blue phenotype on screening plates due to functional complementation of the CyaA fragments (T18 and T25). After 48 h of incubation at 30°C, plates containing around 100 colonies were evaluated. Representative colonies were transferred to liquid LB cultures containing kanamycin sulfate and ampicillin sodium salt and incubated o/n at 30°C. Subsequently, 2 µl of the o/n culture were spotted on LB X-Gal–IPTG screening plates. Pictures were taken after 48 h of cultivation at 30°C.Protein-protein interactions were detected using the bacterial adenylate cyclase two-hybrid system kit (Euromedex) according to the product manuals. Chemically competent  E. coli For quantification of interaction strength, which corresponds to the β-galactosidase activity, cells were inoculated in 1.5 ml LB medium containing 0.5 mM IPTG as well as 50 µg/ml kanamycin sulfate and 100 µg/ml ampicillin sodium salt. After incubation in 2-ml reaction tubes under microaerobic conditions at 30°C for 42 h, cells were harvested and β-galactosidase activities were determined as described above.6-tagged EarPPpu variants (pBAD33-earPPpu) were overproduced in E. coli LMG194 by addition of 0.2% arabinose to exponentially growing cells and subsequent cultivation at 18°C o/n. N-terminally His6-tagged EarP (pACYC-DUET-earPPpu) and His6-SUMO-tagged EF-PPpu (pET-SUMO-efpPpu) were overproduced in E. coli BL21(DE3) by addition of 1 mM IPTG to exponentially growing cells. Subsequently, cells were incubated at 18°C overnight. Rhamnosylated EF-PPpu (EF-PRha) was produced by cooverproduction with His6-tagged EarPPpu. Cells were lysed by sonication, and His6-tagged proteins were purified using Ni-nitrilotriacetic acid  according to the manufacturer’s instructions. The His6-SUMO tag was removed by incubation with 1 μ/mg His6-Ulp1  and 13C-labeled glucose were used. For NMR backbone assignment of EarPPpu, additionally 99.8%-pure heavy water D2O (Sigma-Aldrich) was used instead of H2O in growth medium to allow partial deuteration of the protein in order to reduce cross-relaxation effects and increase the signal-to-noise ratio. Size exclusion chromatography of EarPPpu and the D274AEarP variant was performed in 100 mM NaPi (pH 7.6) 50 mM NaCl using a Superdex 200 Increase 10/300-Gl column with a flow rate of 0.3 ml/min on an Äkta purifier . Four milligrams of protein was loaded in a volume of 0.5 ml (8 mg/ml). Eluting protein was detected at 280 nm. Fractions of 0.5 ml were collected.C-terminally Hisis6-Ulp1  overnighl medium . If necePpu, E. coli BL21(DE3) cells expressing N-terminally His6-tagged EarPPpu were cultivated in 1 liter M9 minimal medium at 37°C to an optical density at 600 nm (OD600) of 0.6. One hundred micrograms of threonine, 100 µg lysine, and 50 µg isoleucine were added to feedback inhibit methionine biosynthesis (l-(+)-selenomethionine was added 15 min prior to induction. Protein production was induced by addition of 1 mM IPTG, and cells were incubated at 18°C overnight. Protein concentrations were determined as described by Bradford , whereas a buffer composed of 100 mM NaPi, pH 7.6, 50 mM NaCl, and 5 mM DTT was used when the proteins were subjected to NMR analysis.For the production of selenomethylated EarPynthesis . AdditioBradford . For bio2Cl2 was distilled from calcium hydride, and tetrahydrofuran (THF) was distilled from sodium benzophenone immediately prior to use. Dimethylformamide (DMF) was stored under argon in a flask containing 4 Å molecular sieves. Reactions were monitored by thin layer chromatography (TLC) with precoated Silica Gel 60 F254 aluminum plates  using UV light and methoxyphenol reagent (100 ml 0.2% ethanolic methoxyphenol solution and 100 ml 2 M ethanolic sulfuric acid) as the visualizing agent. Flash chromatography was performed using silica gel (35 to 70 μm) from Acros Organics. Peptide purification by reverse-phase high-performance liquid chromatography (RP-HPLC) was performed on a JASCO purification system with a UV–visible-light detector (model UV-2075Plus) using a Phenomenex Aeris Peptide 5-μm XB-C18 column (250 by 21.2 mm). Analytical RP-HPLC was measured on a JASCO system with a Phenomenex Aeris Peptide 5-μm XB-C18 column (250 by 4.6 mm). In all cases, mixtures of water (eluent A) and acetonitrile (eluent B) were used as eluents; if required, 0.1% formic acid (FA) or 0.1% trifluoroacetic acid (TFA) was added. High-resolution electrospray ionization (HR-ESI) mass spectra were recorded on a Thermo Finnegan LTQ FT mass spectrometer or on a Bruker maxis apparatus equipped with a Waters ACQUITY ultrahigh-performance liquid chromatograph (UPLC) using a Kinetex C18 column  at 40°C , and 5 eq of N,N′-diisopropylcarbodiimide. All coupling reactions were conducted at 75°C and 28 W for 5 min. Removal of the allyloxycarbonyl-protecting group and subsequent coupling of the sugar moiety, as well as deprotection of the acetyl groups, were performed according to established procedures , calculated for C44H82N14O16 [M+2H]2+, m/z = 531.3011; found, 531.3016.High-resolution mass spectrometry (HRMS) (ESItR (retention time) = 9.61 min, λ = 204 nm , = 204 nm .RhaNAAIVK. To test the specificity of the purified polyclonal antibodies toward EF-PRha, 1.5 µg of unmodified and 0.5 µg of modified EF-P were transferred to a nitrocellulose membrane by Western blotting. While polyclonal antibodies that were raised against EF-P from S. oneidensis detect both unmodified and modified EF-PPpu, anti-ArgRha specifically detects the modified protein variant  program. The mono-rhamnosyl-arginine-containing peptide was coupled to bovine serum albumin (BSA) according to an internal protocol (AS-PECO 05). Antibodies capable of binding to rhamnosyl-arginine were purified from rabbit sera by affinity chromatography (AS-PURI−MED) against the glycopeptide SGR variant .6 tag , 0.2 μg/ml anti-EF-P, or 0.25 μg/ml of anti-ArgRha. Primary antibodies (rabbit) were targeted by 0.2 μg/ml alkaline phosphatase-conjugated anti-rabbit IgG (H&L) (goat) antibody (Rockland). Target proteins were visualized by addition of substrate solution .Electrophoretic separation of proteins was carried out using SDS-PAGE as described by Lämmli . SeparatPpu (0.1 µM) and unmodified EF-PPpu (2.5 µM) constant. A mixture of EarPPpu and unmodified EF-PPpu was equilibrated to 30°C in 100 mM NaPi (pH 7.6). The reaction was started by the addition of TDP-Rha and was stopped after 20 s of incubation at 30°C by the addition of 1 vol of 2× Lämmli buffer (Ppu was detected as described above. Band intensities were quantified using ImageJ (in vivo) rhamnosylated EF-PPpu. Km and kcat values were determined by fitting reaction rates (in nanomoles per milligram per second) to the Michaelis-Menten equation using SigmaPlot. Time course experiments conducted at a TDP-Rha concentration of 500 µM show that the rhamnosylation reaction is not saturated after 20 s of incubation , harvested by centrifugation, and resuspended in 25 ml 100 mM NaPi (pH 7.6) (2 × 1010 cells/ml). After disruption of cells with a Constant Systems Ltd. continuous-flow cabinet at 1.35 kb, cell debris were removed by centrifugation and lysates were sterilized by filtration (Steriflip). A mixture of EarPPpu (0.1 µM) and unmodified EF-PPpu (2.5 µM) was equilibrated to 30°C in 10 µl 100 mM NaPi (pH 7.6). The reaction was started by addition of 10 µl lysate from ~2 × 107 or ~2 × 108 cells and stopped after 20 s of incubation at 30°C by addition of 1 vol 2× Lämmli buffer (Ppu was detected as described above. Band intensities were quantified using ImageJ (3 for E. coli (3 for P. putida and P. aeruginosa (Cells were grown in 1 liter LB to an ODi buffer  and incui buffer . Samplesg ImageJ . TDP-Rha E. coli  and 2.1 ruginosa .All NMR experiments were performed at 298 K on Bruker Avance III spectrometers with a magnetic field strength corresponding to a proton Larmor frequency of 600 MHz (equipped with a Bruker TXI cryogenic probe head), 700 MHz (equipped with a Bruker room temperature probe head), or 800 MHz (equipped with a Bruker TXI cryogenic probe head). All data sets were processed using NMRPipe .15N- and 13C-labeled EF-P (700 µM) in 100 mM NaPi, 50 mM NaCl, and 5 mM DTT (pH 7.6), 0.02% NaN3 was added to the sample. Sequential resonance assignment was obtained from two-dimensional (2D) 1H-15N HSQC and three-dimensional (3D) HNCA, CBCACONH, and HNCACB backbone experiments, using a constant time during 13C evolution (http://cara.nmr.ch) and CcpNmr Analysis (63), and 98% of the backbone resonances could be assigned. Missing assignments for residues other than prolines are S123, R133, N140, V164, D175, and G185. Secondary chemical shift analysis was performed based on the difference between measured 13Cα and 13Cβ chemical shifts and random coil chemical shifts of the same nuclei to assign a secondary structure to the EF-P sequence , enabled backbone resonance assignment of 62% of all assignable residues (excluding prolines).Due to the size of EarP (43 kDa), backbone resonance assignment was possible only for OSY mode . TROSY-HOSY mode , processOSY mode  and anal15N-labeled protein sample and monitoring the progress of the titration by recording 1H-15N HSQC. First, 15N-labeled 150 µM unmodified EF-P was titrated with unlabeled EarP to a 1:2 EF-P/EarP molar ratio with intermediate steps at 1:0, 1:0.5, 1:1, and 1:1.5 EF-P/EarP molar ratios. 15N-labeled 41 µM rhamnosylated EF-P was titrated with unlabeled EarP to a 1:2 EF-P/EarP molar ratio without any intermediate steps. 15N-labeled 540 µM wild-type EarP was titrated with unlabeled TDP-Rha to a 1:5 EarP/TDP-Rha molar ratio with intermediate steps at 1:0, 1:0.2, 1:1, and 1:3 molar ratios. 15N-labeled 186 µM D13A variant or 209 µM D17A EarP variant was titrated by the addition of TDP-Rha to an approximately 1:10 molar ratio with no intermediate steps. To analyze the EF-P/EarP and wild-type EarP/TDP-Rha ratio titration, the chemical-shift perturbations (CSPs) were calculated according to the formula The NMR titrations were always performed by adding an unlabeled interaction partner to the 1H-15N HSQC spectra of 15N-labeled EarP variants with the following single-amino-acid substitutions at the indicated concentrations were recorded: 209 µM D13A, 209 µM D17A, 162 µM F191A, 197 µM Y193A, 139 µM D274A, 186 µM R271A, and 162 µM Y291A.To check proper folding of EarP variants, EarP or mutants and either 70 µM (1:7 ratio of protein to ligand to mimic SAXS conditions) or 1 mM TDP-Rha in 100 mM potassium phosphate buffer, pH 7.5, 150 mM NaCl, 1 mM DTT, and 10% D2O. The experiments were performed on a Bruker Avance III 700-MHz spectrometer equipped with a triple resonance (TXI) room temperature probe head at 277 K. Protein was saturated with 49-ms Gaussian pulses at the resonance frequency of methyl resonances at 0.592 ppm. The experimental results were collected after a total saturation time of 20 s, with 1,596 scans performed for the WTEarP sample with a 100-fold excess of ligand, and after a total saturation time of 5 s, with 4,096 scans performed for the WTEarP sample with a 7-fold excess of ligand. For EarP mutants, the experimental results were collected after a total saturation time of 4 s and with 128 scans.STD NMR experiments were performed with 10 µM WTThirty microliters of EarP, EarP plus TDP-rhamnose, and buffer (with and without TDP-rhamnose) were measured at 20°C at BioSAXS beamline BM29 at the European Synchrotron Radiation Facility using a 2D Pilatus detector. For each measurement, 10 frames with a 1-s exposure time per frame were recorded for each EarP and buffer sample, using an X-ray wavelength (λ) of 0.9919 Å. Measurements were performed in flow mode, where samples are pushed through a capillary at a constant flow rate to minimize radiation damage. The protein concentrations measured were 1.0, 2.0, 4.0, and 8.0 mg/ml. TDP-Rha was used in a 7:1 excess (ligand to protein). The buffer measurements were subtracted from each protein sample, and the low Q range of 1.0 mg/ml was merged with the high Q range of the 8.0-mg/ml sample, using PRIMUS . The mer6-tagged EarPPpu expressed as a seleno-methionine derivative was used. The protein was dialyzed to 50 mM Tris, 100 mM NaCl, 1 mM DTT, pH 7.6, and concentrated to 183 µM. TDP-Rha was added to a final concentration of 10 mM. The crystallization condition was 0.2 M ammonium acetate, 0.1 M bis-Tris (pH 6.0), and 27% (wt/vol) polyethylene glycol 3350. A full data set was collected at the ID29 beamline, ESRF, Grenoble, France, at a wavelength of 0.97 Å (the absorption peak for selenium) and with a 15.05% beam transmission with a 0.15° oscillation range, 0.037-s exposure time, and 2,400 frames. The space group was determined to be I4. The data set was phased using single-wavelength anomalous diffraction (SAD) by the Crank2 (For crystallization, N-terminally Hise Crank2  automatie Crank2 , using Ae Crank2  for subse Crank2 , complete Crank2 , and useAtomic coordinates and structure factors for the reported crystal structures have been deposited with the Protein Data Bank under accession number 5NV8.


This study aimed to evaluate the anti-aging effect of a newly prepared insect-derived compound, dung beetle glycosaminoglycan (GAG), given intraperitoneally to old SD rats as part of their diet for 1 month. Insect GAG administration was found to be related to a reduction in oxidative damage, hepato-cellular biomarker levels, protein carbonyl content, and malondialdehyde concentration. The anti-aging-related molecular genetic mechanisms of dung beetle GAG are not yet fully elucidated.Catharsius molossus (a type of dung beetle) GAG (CaG) possessed anti-aging activities; it reduced the serum level of creatinine kinase, had aortic vasorelaxant activities and cardioprotective actions, and maintained a normal glucose level in treated rats. Microarray analysis was performed with a rat 30 K cDNA clone set array to identify the gene-expression profiles of 14-month-old SD rats treated with dung beetle glycosaminoglycan 5 mg/kg (CaG5) over a 1-month period, which was done to investigate its anti-aging effect as compared to that of either Bombus ignitus (a type of bumblebee) queen GAG 5 mg/kg (IQG5) or chondroitin sulfate 10 mg/kg. CaG5 and IQG5 had marked anti-inflammatory effects, bringing about inhibition of free fatty acid, uric acid, sGPT, IL-1 beta, and CK values. In addition, anticoagulant and antithrombotic effects were seen: the concentration of factor 1 (fibrinogen) was increased in CaG- treated rat plasma. The CaG5-treated rat group, compared to the control, displayed upregulation of 131 genes, including lipocalin 2 (Lbp) and a serine peptidase inhibitor, Kaszal type3 (Spink3), and 64 downregulated genes, including lysyl oxidase (Lox), serine dehydratase (sds), and retinol saturase (Retsat).Our data suggest that dung beetle glycosaminoglycan may be a helpful treatment for aged rats, which indicates its potential as a therapeutic biomaterial for aging. Catharsius molossus, which is found in China, displayed marked fibrinolytic activity and anti-oxidative and anti-hyperglycemic effects on rats fed a high fat diet  of rat liver tissues treated with GAG in a HFD, as assessed by toluidine blue O staining, were reduced by CaG5 (33.00 ± 1.73), IQG5 (45.00 ± 4.36), or CS10 (26.00 ± 2.52)  when compared to the control (51.00 ± 5.29)  Fig. .No significant differences in blood pressure (systolic blood pressure or heart rate) were observed between the 5 mg/kg CaG-, IQG-, or CS-treated groups and the control group (data not shown).vs CON, p < 0.05); IQG5, 627.14 ± 160.67; CS10, 790.00 ± 274.22 . Prothrombin time (sec.) was as follows: CON, 62.86 ± 47.30; CaG5, 52.71 ± 4.96; IQG5, 52.14 ± 5.58; CS10, 55.00 ± 9.92Some dose-dependent changes were observed between the treatment and control groups with respect to the hematological parameters examined at the end of the experiment. There was a 27.5% increase in APTT (sec.) in the CaG5-treated group: CON, 35.68 ± 5.68; CaG5, 45.49 ± 10.91; IQG5, 29.93 ± 3.57; CS10, 36.83 ± 23.95 ; CS10, 259.7 ± 57.27 . Free fatty acid levels (μEq/L) were decreased: CON, 686.78 ± 104.67; CaG5, 431.33 ± 66.22 ; IQG5, 405.6 ± 63.39 ; CS10, 566.9 ± 115.38 . Also, serum GPT (ALT) levels (IU/L) in the CaG- and IQG-treated groups were significantly lower than those in the control group in aged rats: CON, 76.33 ± 40.21; CaG5, 36.78 ± 8.0 IU/L; IQG5, 43.22 ± 13.86 ; CS10, 53.44 ± 11.36 . Furthermore, the mean creatinine phosphokinase (CK U/L) level in the CaG-treated group was lower than that in the control group in aged rats: CON, 125.78 ± 91.66; CaG5, 102.89 ± 65.73 ; IQG5, 91.22 ± 24.74; CS10, 131.2 ± 62.56. Mean serum glucose (mg/dL) was significantly decreased in aged rats : CON, 435.11 ± 105.42; CaG5, 304.0 ± 44.44; IQG5, 275.9 ± 43.18; CS10, 317.8 ± 60.31.In sera from the IQG- and CS-treated groups Table , phosphovs. CON, p < 0.05); CS10, 198.9 ± 44.43.The mean total cholesterol level (mg/dl) in the IQG-treated group was lower than in the control group in aged rats, and that difference was also significant: CON, 212.67 ± 92.82; CaG5, 181.44 ± 68.4; IQG5, 124.1 ± 23.6 ; CS10, 183.4 ± 104.74. LDL cholesterol (mg/dL) levels decreased: CON, 74.67 ± 44.91; CaG5, 69.0 ± 31.3; IQG5, 41.44 ± 10.14 ; CS10, 65.22 ± 18.52. Significant uric acid level (mg/dL) decreases were seen in all GAG-treated groups as compared with the control : CON, 10.9 ± 2.26; CaG5, 8.12 ± 1.48; IQG5, 6.49 ± 0.89; CS10, 8.14 ± 2.17, Table In addition, triglyceride levels (mg/dL) were as follows: CON, 207.89 ± 101.95; CaG5, 136.22 ± 61.6; IQG5, 103.3 ± 35.59  was assayed after 1 month of each GAG treatment [CON, 252.9 ± 13.1; CaG5, 175.1 ± 5.8 (30.32% decreases). Each GAG and chondroitin sulfate treatment decreased the lipid peroxidation in hepatocytes Fig. . The prop < 0.05), and glutathione s-transferase activity (nmol/min/ml) in the CaG group was also significantly increased compared to that in the control group   in hepatocytes after 1 month of GAG treatment was as follows: CON, 17.72 ± 2.81; CaG5, 19.87 ± 3.88; IQG5, 17.22 ± 4.55; CS10, 19.23 ± 3.55. Catalase activity in all CaG-treated hepatocyte groups increased. Super oxide dismutase (SOD) is a free radical (super oxide) scavenging enzyme. SOD activity (nmol/min/ml) increased in the treatment groups as compared with the control: control, 381.44 ± 85.32; CaG5, 400.56 ± 60.62; IQG5, 386.72 ± 69.22; CS10, 513.05 ± 60.07. Glutathione peroxidase activity (unit/mg protein) in the CaG-treated hepatocyte group was significantly increased by the treatment . IL-10 activity (ρg/ml) after 1 month of CaG treatment in rats was increased: CON, 34.9 ± 11.5; CaG5, 67.4 ± 13.3 ; IQG5, 55.5 ± 24.7; CS10, 28.0 ± 3.8 .Increases in IL-10 levels were observed in the CaG-treated group. IL-1β (ρg/ml) levels in serum were reduced by 1 month of CaG or IQG treatment, demonstrating the compounds’ anti-inflammatory actions: control, 251.3 ± 70.7; CaG5, 102.9 ± 9.5; IQG5, 110.4 ± 5.2; CS10, 110.4 ± 10.1  when concentration of PCR amplicon are equilibrated by sample amplicon. Median CT values of 5 sample’s represented, but there was no different from control group  on used primer sequence (data not shown).Microarray analysis using a Mouse 28 K cDNA clone array was performed in order to identify the gene-expression profiles in CaG, IQG, and CS-treated 15-month-old SD rat livers, and to provide information on potential anti-aging markers.Compared to the control group, the CaG5 group exhibited a 2- to 4-fold increase in 118 genes, a 4- 8-fold increase in 11 genes, and a greater than 8-fold increase in 2 genes. The expression levels of the remaining ~30,000 genes remained the same. Sixty genes were downregulated from 2- to 4-fold, and 4 showed a 4- to 8-fold decrease.In the IQG5 group as compared to the control group, 162 genes increased by 2- to 4-fold, 10 genes increased by 4- to 8-fold, and 3 genes increased by more than 8-fold. On the other hand, 66 were downregulated by 2- to 4-fold and 7 were reduced by 4- to 8-fold.In the CS10 group as compared to the control group 63 genes were upregulated by 2- to 4-fold, 2 genes increased by 4- to 8-fold, and 2 genes were upregulated by more than 8-fold. Thirty-eight genes were downregulated by 2- to 4-fold, 4 were downregulated by 4- to 8-fold, and 4 decreased by more than 8-fold.When compared to the control group, the lipocalin2 (Lcn2) gene was increased by about 10-fold in CaG5-treated liver tissue, 46-fold in the IQ5 group, and 23-fold in the CS10 group. In liver tissues of CaG5-treated mice, lysil oxidase (Lox) was downregulated at a 0.13 ratio, that is, gene expression was decreased. CaG5 treated rat group, compared to control, showed that 131 genes including lipocalin 2, liposaccharide binding protein (Lbp), and serine peptidase inhibitor, Kaszal type3 (Spink3) were up-regulated  treatment, showing that heart stimulation occurred in a concentration-dependent manner , showing COX-1 and COX-2 inhibitory activity [Bombus ignitus) extract was shown to contain bee venom serine protease [Aging is a process of progressive decline in the physiological capacity of an organism, manifested by accumulated alterations and destabilization at the whole system level . Glycosaactivity ; chitosaactivity ; melaninactivity ; and seractivity , 28. A bprotease , peroxirprotease , and pepprotease .Strategies to prevent or delay aging have included ingestion of antioxidants for repairing oxidative cellular damage to DNA, proteins, and lipids, as well as stem cell-mediated tissue regeneration and gene therapy . As a liC. molossus and B. ignitus queens consist mainly of D-glucosaminic acid as an acidic monosaccharide and N-acetyl-galactosaminitol as an amino monosaccharide, along with α-glucose and D-mannitol as neutral monosaccharides. Thus, the compound can access cell membrane receptors and can prevent the causes of age-related diseases by altering energy metabolism. Glycosaminoglycans could repair connective tissues [Glycosaminoglycans from  tissues , eroded  tissues . In thisData generated from the DNA microarray analysis supported the anti-aging effect of IQG, with meaningful changes in gene expression profiles in 14-month-old SD rats observed after a 1-month treatment period. The CaG5-treated rats, compared to the control group, had increases in 131 genes, including lipocalin 2 , liposacThe present study observed meaningful changes in gene expression profiles in aged rats after treatment with CaG or IQG for 1 month. The gene expression of lipocalin-2, which is an adipokine implicated in insulin resistance  and a seDung beetle glycosaminoglycan decreased cholesterol and triglyceride levels in serum, caused a decrease in the weight of adipose tissue and a normalization of serum levels of free fatty acid, GPT, glucose, and uric acid, and a prolongation of coagulation time that would prevent blood aggregation and lipid accumulation in vascular endothelial barriers that contribute to homeostasis in the circulatory system. IL-1β (pg/ml) in serum was reduced by 1 month of CaG or IQG treatment, demonstrating anti-inflammatory action. In addition, the liver protective gene lipocalin was highly (10-fold) upregulated and lysyl oxidase was downregulated (0.1-fold) in CaG-treated rats. CaG derived from insects could be a safer replacement for heparin and other GAGs from mammalian sources, as insect sources reduce the possibility of transmission of infectious viruses from animals such as pigs. These results suggest that CaG and BIQ could not only be natural anti-aging agents but also be functional foods, similar to chitosan and future biomaterials playing vital role our to-day to day life.


Centella asiatica (C. asiatica) possesses antioxidant, anti-inflammatory and anti-tumour potential. We investigated the modulation of antioxidants, cytokines and cell death by C. asiatica ethanolic leaf extract (CLE) in leukaemic THP-1 cells and normal peripheral blood mononuclear cells (PBMC’s).Cancer cachexia is associated with increased pro-inflammatory cytokine levels. LE was determined at 24 and 72 h (h). Oxidant scavenging activity of CLE was evaluated using the 2, 2-diphenyl-1 picrylhydrazyl (DPPH) assay. Glutathione (GSH) levels, caspase  activities and adenosine triphosphate (ATP) levels (Luminometry) were then assayed. The levels of tumour necrosis factor-α (TNF-α), interleukin (IL)-6, IL-1β and IL-10 were also assessed using enzyme-linked immunosorbant assay.Cytotoxcity of CLE decreased PBMC viability between 33.25–74.55% (24 h: 0.2–0.8 mg/ml CLE and 72 h: 0.4–0.8 mg/ml CLE) and THP-1 viability by 28.404% (72 h: 0.8 mg/ml CLE) (p < 0.0001). Oxidant scavenging activity was increased by CLE (0.05–0.8 mg/ml) (p < 0.0001). PBMC TNF-α and IL-10 levels were decreased by CLE (0.05–0.8 mg/ml) (p < 0.0001). However, PBMC IL-6 and IL-1β concentrations were increased at 0.05–0.2 mg/ml CLE but decreased at 0.4 mg/ml CLE (p < 0.0001). In THP-1 cells, CLE (0.2–0.8 mg/ml) decreased IL-1β and IL-6 whereas increased IL-10 levels (p < 0.0001). In both cell lines, CLE  increased GSH concentrations (p < 0.0001). At 24 h, caspase  activities was increased by CLE (0.05–0.8 mg/ml) in PBMC’s whereas decreased by CLE (0.2–0.4 mg/ml) in THP-1 cells (p < 0.0001). At 72 h, CLE (0.05–0.8 mg/ml) decreased caspase  activities and ATP levels in both cell lines (p < 0.0001).CLE proved to effectively modulate antioxidant activity, inflammatory cytokines and cell death. In THP-1 cells, CLE decreased pro-inflammatory cytokine levels whereas it increased anti-inflammatory cytokine levels which may alleviate cancer cachexia.In PBMC’s and THP-1 cells, C The role of inflammation in carcinogenesis has been extensively documented . AlthougMany malignancies are associated with the cachectic syndrome , a disorOxidative stress, inflammatory cytokines and apoptosis play a pivotal role in the initiation and development of cancer cachexia . InflammOxidative stress has been associated with tumour initiation, inflammation , 3 and mCentella asiatica (Linnaeus) Urban (C. asiatica) is native to India, China, and South Africa , South Africa (SA) and identified by Dr. Kathleen Immelman from the Department of Botany at the Walter Sisulu University, SA. Voucher specimens were deposited at the KEI herbarium (13979). The THP-1 cells were obtained from American Type Culture Collection . RPMI-1640 and BD OptEIA enzyme-linked immunosorbant assay (ELISA) cytokine kits were purchased from The Scientific Group . Foetal calf serum (FCS) and Pen/Strep Amphotericin B (PSF) were acquired from Whitehead Scientific . Dimethyl sulphoxide (DMSO) was purchased from Merck . Histopaque-1077, LPS and 2, 2-diphenyl-1 picrylhydrazyl (DPPH) were purchased from Sigma . The 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulphonate (WST-1) cell proliferation reagent was purchased from Roche . Promega  supplied the caspase , adenosine triphosphate (ATP) and glutathione (GSH) kits.Centella asiatica (L.) Urb and has been confirmed by using the plant list  serially . Plates were washed (3×), standards and samples  were aliquoted into appropriate wells and plates were incubated . Plates were washed (5×), working detector (100 μl/well) added and plates incubated . The plates were washed (7×), substrate solution (100 μl/well) added and plates were incubated  in the dark. Finally, stop solution (50 μl/well) was added and the absorbance was read at 450 nm (570 nm reference wavelength) with a Multiskan FC micro-plate reader (Thermo Scientific). Cytokine concentrations were calculated by extrapolation from a standard curve.5 cells/ml) and standards were added into an opaque 96-well plate (duplicate wells), followed by GSH-Glo™ reagent (25 μl/well) and allowed to incubate  in the dark. Subsequently, luciferin detection reagent (50 μl/well) was added and plates incubated  in the dark. The absorbance was read on a Modulus™ microplate luminometer  and GSH concentrations were calculated by extrapolation from a standard curve.The GSH-Glo™ assay  was used to measure GSH levels. Standard GSH solutions were prepared by diluting a 5 mM stock solution serially (1.56–50 μM) and PBS (0.1 M) was the standard blank. Cells  were added into duplicate wells of a microtitre plate for each assay, thereafter caspase −3/7, −8, −9 and ATP reagents (100 μl/well) were added into appropriate wells. The plate was incubated  in the dark. Luminescence was measured on a Modulus™ microplate luminometer (Turner BioSystems) and expressed as relative light units (RLU).Caspase activity and ATP levels were determined using the Caspase-Glo®-3/7, −8, −9 and ATP assay kits . Caspase-Glo®-3/7, −8, −9 and ATP reagents were reconstituted according to the manufacturer’s instructions. Cells  statistical analysis software. The one-way analysis of variance (ANOVA) was used to make comparisons between groups, followed by the Tukey multiple comparisons test, with LE using the DPPH assay is shown in Fig. LE (0.05–0.8 mg/ml) significantly increased DPPH scavenging activity by approximately 45–84% .The oxidant scavenging activity of CLE  dose dependently decreased PBMC viability by 33.25–61.85% , whereas THP-1 viability was not significantly altered as compared to the control . At 72 h, CLE decreased both PBMC , suggesting that PBMC’s are more sensitive to CLE treatment than THP-1 cells.The WST-1 assay was used to determine cell viability of THP-1 cells and PBMC’s after treatment with CLE Fig. . At 24 hLE altered cytokine levels in PBMC’s and THP-1 cells which are shown in Figs. LE (0.05–0.2 mg/ml) increased PBMC IL-1β and IL-6 concentrations relative to the control . In PBMC’s, TNF-α, IL-1β and IL-6 concentrations were decreased at 0.05–0.8 mg/ml CLE, 0.4–0.8 mg/ml CLE and 0.4 mg/ml CLE respectively as compared to the control . The levels of TNF-α, IL-1β, IL-6 and IL-10 produced in LPS stimulated THP-1 cells was 5.96, 25.92, 98.63, and 2.46 pg/ml respectively. TNF-α concentration in THP-1 cells was increased by CLE  relative to the control. In THP-1 cells, IL-1β and IL-6 concentrations were increased by 0.05 mg/ml CLE whereas decreased by 0.2–0.8 mg/ml CLE as compared to the control . Concentration of the anti-inflammatory cytokine, IL-10 was decreased in PBMC’s while increased in THP-1 cells by CLE (0.05–0.8 mg/ml) relative to the control .CLE was determined by measuring GSH levels in both cell lines (Table LE but decreased by 0.4–0.8 mg/ml CLE relative to the control (Table p < 0.0001). In THP-1 cells, CLE (0.05–0.8 mg/ml) increased GSH levels as compared to the control (Table p < 0.0001). At 24 h, GSH concentrations were increased to a greater extent in THP-1 cells (0.068–3.890 μM) than PBMC’s (0.191–1.746 μM). At 72 h, CLE (0.05–0.8 mg/ml) increased GSH concentrations in PBMC’s and THP-1 cells by 1.13–5.91 μM and 0.12–0.19 μM respectively as compared to the control (Table p < 0.0001). Notably, CLE increased GSH levels to a greater extent in PBMC’s as compared to THP-1 cells at 72 h.The endogenous antioxidant activity of Ces Table . At 24 hLE. The pro-apoptotic effect of CLE in PBMC’s treated for 24 h is shown in Table LE, whereas decreased by 0.4–0.8 mg/ml CLE as compared to the control (Table p < 0.0001). CLE  increased PBMC caspase −9 and −3/7 activities relative to the control (Table p < 0.0001). Increased caspase activity led to the initiation and execution of PBMC apoptosis at 24 h. The PBMC ATP levels were increased by 0.4 mg/ml CLE, whereas decreased by 0.05, 0.2 and 0.8 mg/ml CLE (Table p < 0.0001).Luminometry assays were used to determine caspase activity and ATP levels in THP-1 cells and PBMC’s after treatment with CLE pro-apoptotic effects in THP-1 cells treated for 24 h is shown in Table LE (0.05–0.8 mg/ml) increased THP-1 caspase-8 activity as compared to the control (Table p < 0.0001). In THP-1 cells, caspase-9 activity and ATP levels were decreased by 0.05–0.4 mg/ml CLE, whereas increased by 0.8 mg/ml CLE relative to the control (Table p < 0.0001). The THP-1 caspase-3/7 activity was decreased by 0.2–0.4 mg/ml CLE, whereas increased by 0.05 and 0.8 mg/ml CLE as compared to the control (Table p < 0.0001). THP-1 caspase  activities was increased by 0.8 mg/ml CLE, suggesting an increased initiation and execution of THP-1 apoptosis.CLE in PBMC’s treated for 72 h is shown in Table LE, whereas decreased by 0.05, 0.2, 0.8 mg/ml CLE relative to the control (Table p < 0.0001). CLE (0.05–0.8 mg/ml) decreased PBMC caspase  activities and ATP levels as compared to the control (Table p < 0.0001). Decreased PBMC caspase activity suggests a decrease in PBMC apoptotic cell death.The pro-apoptotic effect of CLE pro-apoptotic effects in THP-1 cells treated for 72 h is shown in Table LE whereas decreased by 0.05, 0.2, 0.8 mg/ml CLE relative to the control (Table p < 0.0001). CLE (0.05–0.8 mg/ml) decreased THP-1 caspase  activities and ATP levels as compared to the control (Table p < 0.0001). Decreased THP-1 caspase activity suggests a decrease in THP-1 apoptotic cell death.CC. asiatica possesses anti-inflammatory  and tumour progression by promoting angiogenesis and metastasis [LE to increase pro-inflammatory cytokines such as IL-1β in PBMC’s may aid in cancerous cell elimination through increased host anti-tumour activity. Conversely, in THP-1 cells, the decrease in IL-6 and IL-1β concentrations by CLE may diminish cytokine induced tumour immunosuppressive activity and cancer progression.Inflammatory cytokines play an essential role in tumourgenesis and the cachectic syndrome . Previourophages . Our resβ levels . Our fintastasis . Previoutastasis . In additastasis . The abiLE (0.4 mg/ml) to decrease IL-6 and IL-1β concentrations in PBMC’s and THP-1 cells suggests a decrease in LPL inhibition and HSL stimulation, thus contributing to lipogenesis maintenance and minimal lipolysis. IL-6 and TNF-α further contribute to cachexia by stimulating muscle catabolism through the activation of the ubiquitin-proteasome pathway [LE (0.4 mg/ml) may prevent excessive activation of NF-κB and proteasome pathways, ultimately decreasing proteolysis. Taken together, CLE may be able to decrease tissue wasting through the down regulation of pro-inflammatory cytokine levels.With regard to the cachectic syndrome, TNF-α inhibits the production of LPL and reduces the rate of LPL gene transcription –42, ther pathway , 22, 44. pathway . Taken t pathway . In cach pathway , thereby pathway . By decrLE to increase IL-10 levels will facilitate a decrease in pro-inflammatory cytokine levels, a decrease in malignant cell progression and possibly alleviate the cancer cachectic syndrome.The immunosuppressive and anti-inflammatory cytokine IL-10, inhibits tumour development, tumour progression, modulates apoptosis and suppresses angiogenesis during tumour regression . AdditioLE increased GSH concentrations. At 72 h, CLE (0.4 mg/ml) increased GSH levels more significantly in PBMC’s (1.45-fold) than THP-1 cells (1.11-fold). This suggests that CLE induces a higher antioxidant defense in normal PBMC’s than cancerous THP-1 cells at 72 h.GSH, a potent antioxidant , effectiApoptosis is a tightly regulated process involving a number of check points before an irreversible point is reached . The extLE  decreased caspase-8 activity. An increase in TNF-α levels initiates the extrinsic apoptotic pathway subsequently activating caspase-8. However, CLE decreased PBMC TNF-α levels which may have contributed to the decreased caspase-8 activity. At 24 h, CLE increased PBMC caspase (−8 (0.05–0.2 mg/ml), −9, −3/7 (0.05–0.8 mg/ml)) activities, suggesting the activation of the extrinsic and intrinsic apoptotic pathways. GSH regulates apoptosis by preventing ROS accumulation [LE  may have increased apoptosis ultimately decreasing PBMC cell viability. In THP-1 cells, CLE (0.05–0.4 mg/ml) increased caspase-8 activity and decreased caspase-9 activity, suggesting initiation of apoptosis through the extrinsic pathway (24 h). In CLE treated THP-1 cells, the decreased caspase-9 activity may have been a consequence of the increased GSH levels. Although extrinsic apoptosis was activated in THP-1 cells, CLE (0.2–0.4 mg/ml) decreased caspase-3/7 activity, indicating that apoptosis was not fully executed (24 h). Interestingly, CLE increased THP-1 caspase  activities at 0.8 mg/ml (24 h), suggesting an increased initiation and execution of THP-1 apoptosis.Previous studies reported that asiatic acid decreased cell viability, induced apoptosis and DNA fragmentation , 52. In mulation . Previoumulation , 55. In LE  may have decreased apoptotic cell death. However, PBMC and THP-1 cell viability was deceased at 0.4–0.8 mg/ml CLE and 0.8 mg/ml CLE respectively, suggesting an alternative form of cell death occurred.At 72 h, caspase activities were decreased in both cell lines, suggesting a decreased activation of apoptosis. In PBMC’s and THP-1 cells, the increase in GSH levels and the decrease in caspase  activities by CLE to down regulate caspase activities in PBMC’s and THP-1 cells may decrease proteolysis and the progression of cancer cachexia.Increased caspase-3 and proteasome activity, as well as E3 ubiquitin-conjugating enzyme expression are associated with increased proteolysis . Thus thLE decreased ATP levels, a possible consequence of the decreased cell viability. Cancer cells require high levels of ATP for cellular proliferation [LE decreased ATP levels which may decrease THP-1 cell proliferation. However in cachexia, a decrease in ATP levels may contribute to tissue wasting.The cachectic syndrome is characterized by a negative energy balance due to reduced food intake and abnormal metabolism . The inaferation . In THP-LE to decrease pro-inflammatory cytokine levels may increase food intake, decrease energy expenditure and possibly combat the negative energy balance associated with cancer cachexia.The potent feeding stimulant neuropeptide Y (NPY) promotes food and energy intake . IncreasLE increased oxidant scavenging activity and GSH levels, modulated pro-inflammatory cytokine levels and regulated apoptosis and caspase activity in normal PBMC’s and THP-1 cells. CLE may thus be effective in cancer cachexia.Our results show that C