Design and synthesis of N-terminal cyclic motilin partial peptides: a novel pure motilin antagonist

Motilin antagonist was designed and synthesized on the basis of the structure-activity relationship analysis of porcine motilin that we reported recently. The drug design was performed on a specific concept to reduce a flexibility of peptide conformation of porcine motilin partial peptide by its cyclization. The cyclic peptide was synthesized using Boc (tert-butyloxycarbonyl) solid phase methodology, followed by cyclization using the azide procedure, and tested for the binding activity to motilin receptor and smooth muscle contractile activity. The cyclic peptides 3, 4, and 5 showed antagonistic property on contraction assay (pA2 [the negative logarithm of molar concentration of antagonist causing a 2-hold shift to the right of the concentration-response curve for motilin]: 4.5, 4.34, and 4.04, respectively, in rabbit duodenum) and no contractile activity even at high concentration.     

氨基酸突变对GABAa受体结合能力的影响

采用同源建模技术构建了大鼠γ-氨基丁酸a型受体(GABAaR)模型, 并将氨基酸残基β157Tyr和β205Tyr突变为相应的突变受体模型. 使用分子对接方法计算了γ-氨基丁酸(GABA)与突变前后受体的相互作用. 对接计算结果显示, Tyr突变为Phe后, 两种突变受体的对接能量大幅提高, GABA生物活性降低; 当Phe的对位引入氟原子后, 对接能量与未突变受体相比更低. 另外, 与β205Tyr突变相比, 与配体距离较近的β157Tyr突变, 对受体与配体作用的影响更大.     

Amino acids of ricin and its polypeptides

Ricin and its corresponding polypeptides (A & B chain) were purified from castor seed. The molecular weight of ricin subunits were 29,000 and 28,000 daltons. The amino acids in ricin determined were Asp45 The22 Ser40 Glu53 Cys4 Gly96 His5 Ile21 Leu33 Lys20 Met4 Phe13 Pro37 Tyr11 Ala45 Val23 Arg20 indicating that ricin contains approximately 516 amino acid residues. The amino acids of the two subunits of ricin A and B chains were Asp23 The12 Ser21 Glu29 Cys2 Gly48 His3 Ile12, Leu17 Lys10 Met2 Phe6 Pro17 Tyr7 Ala35 Val13 Arg13 while in B chain the amino acids were Asp22 The10 Ser19 Glu25 Cys2 Gly47 His1 Ile10, Leu15 Lys11 Met1 Phe7 Pro6 Tyr5 Ala32Val11 Arg10. The total helical content of ricin came around 53.6% which is a new observation.     

Effective synthesis of cyclic peptide yunnanin C and analogues via Ser/Thr ligation (STL)-mediated peptide cyclization

Cyclic peptide yunnanin C isolated from the root of Stellaria yunnanensis was efficiently synthesized in which the linear peptide was prepared by Boc-SPPS and the cyclization was realized by serine/threonine ligation (STL)-mediated cyclization. In addition, nine yunnanin C analogues, including mutations of Tyr7Gly, Tyr7Val, Tyr7Pro, Tyr7Phe, Ser1Thr, Pro2Val, Gly5Pro, Phe6Ala and Ile4Ala, were prepared in the same fashion. Here, we demonstrated that STL-mediated peptide cyclization could be an effective approach to construct cyclic peptides. Except that proline at the C-terminus could retard the cyclization process, cyclization of yunnanin C analogues with various C-terminal amino acids proceeded with fast cyclization rate (<4 h) and only trace amount of dimers (<5%) at a working concentration of 5 mM.     

Putative One‐Pot Prebiotic Polypeptides with Ribonucleolytic Activity

KIA7, a peptide with a highly restricted set of amino acids (Lys, Ile, Ala, Gly and Tyr), adopts a specifically folded structure. Some amino acids, including Lys, Ile, Ala, Gly and His, form under the same putative prebiotic conditions, whereas different conditions are needed for producing Tyr, Phe and Trp. Herein, we report the 3D structure and conformational stability of the peptide KIA7H, which is composed of only Lys, Ile, Ala, Gly and His. When the imidazole group is neutral, this 20‐mer peptide adopts a four‐helix bundle with a specifically packed hydrophobic core. Therefore, one‐pot prebiotic proteins with well‐defined structures might have arisen early in chemical evolution. The Trp variant, KIA7W, was also studied. It adopts a 3D structure similar to that of KIA7H and its previously studied Tyr and Phe variants, but is remarkably more stable. When tested for ribonucleolytic activity, KIA7H, KIA7W and even short, unstructured peptides rich in His and Lys, in combination with Mg⁺⁺, Mn⁺⁺ or Ni⁺⁺ (but not Cu⁺⁺, Zn⁺⁺ or EDTA) specifically cleave the single‐stranded region in an RNA stem–loop. This suggests that prebiotic peptide–divalent cation complexes with ribonucleolytic activity might have co‐inhabited the RNA world.     

Two New Cyclic Tetrapeptides of Streptomyces rutgersensis T009 Isolated from Elaphodus davidianus Excrement

Two new cyclic tetrapeptides, cyclo(l ‐Val‐l ‐Leu‐l ‐Val‐l ‐Ile) ( 1 ) and cyclo(l ‐Leu‐l ‐Leu‐l ‐Ala‐l ‐Ala) ( 2 ), and 15 known compounds, cyclo(Gly‐l ‐Leu‐Gly‐l ‐Leu) ( 3 ), cyclo(l ‐Ser‐l ‐Phe) ( 4 ), cyclo(l ‐Leu‐l ‐Ile) ( 5 ), cyclo(l ‐Tyr‐l ‐Phe) ( 6 ), cyclo(Gly‐l ‐Trp) ( 7 ), cyclo(l ‐Leu‐l ‐Tyr) ( 8 ), cyclo(Gly‐l ‐Phe) ( 9 ), cyclo(l ‐Phe‐trans‐4‐hydroxy‐l ‐Pro) ( 10 ), cyclo(l ‐Leu‐l ‐Leu) ( 11 ), cyclo(l ‐Val‐l ‐Phe) ( 12 ), cyclo(l ‐Val‐l ‐Leu) ( 13 ), cyclo(l ‐Ile‐l ‐Ile) ( 14 ), cyclo(l ‐Tyr‐l ‐Tyr) ( 15 ), turnagainolide A ( 16 ), and bacimethrin ( 17 ) were isolated from the fermentation broth of Streptomyces rutgersensis T009 obtained from Elaphodus davidianus excrement. Their structures were identified on the basis of spectroscopic analysis. Meanwhile, the absolute configurations of the amino acid residues of compounds 1 and 2 were determined by advanced Marfey method. Compound 3 was obtained from a natural source for the first time. The X‐ray single crystal diffraction data of bacimethrin ( 17 ) were also reported for the first time. Compounds 1  –  17 exhibited no antimicrobial activities with the MICs > 100 μg/ml.     

Agonistic and antagonistic activities of neuromedin U-8 analogs substituted with glycine or D-amino acid on contractile activity of chicken crop smooth muscle preparations.

To study the structure-activity relationships of neuromedin U-8 (NMU-8) (H-Tyr-Phe-Leu-Phe-Arg-Pro-Arg-Asn-NH2) and to develop a NMU-8 antagonist, twenty-three NMU-8 analogs substituted with Gly or the corresponding D-amino acid(s) at positions 1-8 were synthesized by solid-phase techniques. On isolated chicken crop preparations, the contractile activity of the synthetic NMU-8 analogs was compared with that of NMU-8 and their antagonistic activity was assayed against NMU-8. The replacement of Phe2, Phe4, Arg5, Pro6, Arg7 or Asn8 with Gly brought about a drastic decrease of the agonistic activities. Substitution of the corresponding D-amino acid residue for Phe2, Phe4, Arg5, Pro6 or Asn8 caused a marked decrease of the agonistic activities, while the replacement of Tyr1 with D-form enhanced the activity. It was further revealed that [D-Pro6]-NMU-8 and [D-Leu3, D-Pro6]-NMU-8 exerted a non-competitive antagonistic activity against NMU-8 with x values of 5.22 +/- 0.12 and 5.34 +/- 0.09, respectively. [D-Phe2, D-Pro6]-NMU-8, [D-Arg5, D-Pro6]-NMU-8 and [D-Pro6, D-Asn8]-NMU-8 showed a very weak antagonism. The results indicated that 1) the side chain of each amino acid at positions 2, 4, 5, 6, 7 and 8 of NMU-8 is of relative importance for the expression of the contractile activity, and 2) [D-Pro6]-NMU-8 and its four analogs acted as an antagonist against NMU-8.     

Reversed-phase HPLC of peptides: Assessing column and solvent selectivity on standard, polar-embedded and polar endcapped columns

We desired to evaluate the chromatographic selectivity for peptides of silica-based RP high-performance liquid chromatography stationary phases with various modifications (polar embedding and polar endcapping on C(18) columns; ether-linked phenyl column with polar endcapping) compared with n-alkyl (C(18), C(8)) and aromatic phenylhexyl columns. Thus, we have designed and synthesized two series of synthetic peptide standards with the sequence Gly-Gly-Leu-Gly-Gly-Ala-Leu-Gly-X-Leu-Lys-Lys-amide, where the N-terminal either contains a free α-amino group (AmC series) or is N(α)-acetylated (AcC series) and where position X is substituted by Gly, Ala, Val, Ile, Phe or Tyr. These represent series of peptides with single substitutions of n-alkyl (Gly相似文献   

Fabrication of nanofibers with uniform morphology by self-assembly of designed peptides

Fabrication of controlled peptide nanofibers with homogeneous morphology has been demonstrated. Amphiphilic beta-sheet peptides were designed as sequences of Pro-Lys-X(1)-Lys-X(2)-X(2)-Glu-X(1)-Glu-Pro. X(1) and X(2) were hydrophobic residues selected from Phe, Ile, Val, or Tyr. The peptide FI (X(1)=Phe; X(2)=Ile) self-assemble into straight fibers with 80-120 nm widths and clear edges, as examined by transmission electron microscopy (TEM) and atomic force microscopy (AFM). The fiber formation is performed in a hierarchical manner: beta-sheet peptides form a protofibril, the protofibrils assemble side-by-side to form a ribbon, and the ribbons then coil in a left-handed fashion to make up a straight fiber. These type of fibers are formed from peptides possessing hydrophobic aromatic Phe residue(s). Furthermore, a peptide with Ala residues at both N and C termini does not form fibers (100 nm scale) with clear edges; this causes random aggregation of small pieces of fibers instead. Thus, the combination of unique amphiphilic sequences and terminal Pro residues determine the fiber morphology.     

Effectiveness of the suzuki-miyaura cross-coupling reaction for solid-phase peptide modification

The Suzuki-Miyaura (SM) cross-coupling reaction has recently become one of the most efficient methods for C-C bond construction opening a wide range of opportunities in organic synthesis. This study focused on the evaluation of the use of the SM reaction to modify peptides using a solid-phase synthesis approach, an avenue that was still not investigated intensively. We used as a peptide model [Ala (1,2,3), Leu (8)]Enk linked to a polystyrene support on which it was previously assembled. The aromatic residues Tyr (4) and Phe (7) of [Ala (1,2,3), Leu (8)]Enk were respectively substituted with p-iodo-Phe, and an SM-related strategy was developed. Results indicated that the reaction conditions involving K 3PO 4 or Na 2CO 3 (base), DMF (solvent), Pd(PPh 3) 4 (catalyst), and temperatures ranging from 50 to 80 degrees C during 20 h were found as optimal. Finally applying those optimal conditions, a series of [Ala (1,2,3), Leu (8)]Enk analogs modified at Tyr (4) or Phe (7) positions was synthesized using diverse boronic acid derivatives.     

The role of cation-pi interactions in biomolecular association. Design of peptides favoring interactions between cationic and aromatic amino acid side chains.

Cation-pi interactions between amino acid side chains are increasingly being recognized as important structural and functional features of proteins and other biomolecules. Although these interactions have been found in static protein structures, they have not yet been detected in dynamic biomolecular systems. We determined, by (1)H NMR spectroscopic titrations, the energies of cation-pi interactions of the amino acid derivative AcLysOMe (1) with AcPheOEt (2) and with AcTyrOEt (3) in aqueous and three organic solvents. The interaction energy is substantial; it ranges from -2.1 to -3.4 kcal/mol and depends only slightly on the dielectric constant of the solvent. To assess the effects of auxiliary interactions and structural preorganization on formation of cation-pi interactions, we studied these interactions in the association of pentapeptides. Upon binding of the positively-charged peptide AcLysLysLysLysLysNH(2) (5) to the negatively-charged partner AcAspAspXAspAspNH(2) (6), in which X is Leu (6a), Tyr (6b), and Phe (6c), multiple interactions occur. Association of the two pentapeptides is dynamic. Free peptides and their complex are in fast exchange on the NMR time-scale, and 2D (1)H ROESY spectra of the complex of the two pentapeptides do not show intermolecular ROESY peaks. Perturbations of the chemical shifts indicated that the aromatic groups in peptides 6b and 6c were affected by the association with 5. The association constants K(A) for 5 with 6a and with 6b are nearly equal, (4.0 +/- 0.7) x 10(3) and (5.0 +/- 1.0) x 10(3) M(-)(1), respectively, while K(A) for 5 with 6c is larger, (8.3 +/- 1.3) x 10(3) M(-)(1). Molecular-dynamics (MD) simulations of the pentapeptide pairs confirmed that their association is dynamic and showed that cation-pi contacts between the two peptides are stereochemically possible. A transient complex between 5 and 6 with a prominent cation-pi interaction, obtained from MD simulations, was used as a template to design cyclic peptides C(X) featuring persistent cation-pi interactions. The cyclic peptide C(X) had a sequence in which X is Tyr, Phe, and Leu. The first two peptides do, but the third does not, contain the aromatic residue capable of interacting with a cationic Lys residue. This covalent construct offered conformational stability over the noncovalent complexes and allowed thorough studies by 2D NMR spectroscopy. Multiple conformations of the cyclic peptides C(Tyr) and C(Phe) are in slow exchange on the NMR time-scale. In one of these conformations, cation-pi interaction between Lys3 and Tyr9/Phe9 is clearly evident. Multiple NOEs between the side chains of residues 3 and 9 are observed; chemical-shift changes are consistent with the placement of the side chain of Lys3 over the aromatic ring. In contrast, the cyclic peptide C(Leu) showed no evidence for close approach of the side chains of Lys3 and Leu9. The cation-pi interaction persists in both DMSO and aqueous solvents. When the disulfide bond in the cyclic peptide C(Phe) was removed, the cation-pi interaction in the acyclic peptide AC(Phe) remained. To test the reliability of the pK(a) criterion for the existence of cation-pi interactions, we determined residue-specific pK(a) values of all four Lys side chains in all three cyclic peptides C(X). While NOE cross-peaks and perturbations of the chemical shifts clearly show the existence of the cation-pi interaction, pK(a) values of Lys3 in C(Tyr) and in C(Phe) differ only marginally from those values of other lysines in these dynamic peptides. Our experimental results with dynamic peptide systems highlight the role of cation-pi interactions in both intermolecular recognition at the protein-protein interface and intramolecular processes such as protein folding.     

非衍生化毛细管区带电泳直接紫外检测法测定茶叶中的4种氨基酸

采用非衍生化毛细管区带电泳直接紫外检测法同时分离测定精氨酸(Arg)、色氨酸(Trp)、苯丙氨酸(Phe)和酪氨酸(Tyr)4种氨基酸,并应用于不同发酵过程的茶叶样品的测定。在分离电压为20 kV、柱温为25 ℃、检测波长为190 nm条件下,以25 mmol/L硼酸-硼砂缓冲溶液(pH 10.0)为运行缓冲液,4种组分在8 min内达到基线分离,Arg、Trp、Phe、Tyr的检出限分别为5.0,1.0,0.3和0.5 mg/L。7次平行测定中,4种组分迁移时间的相对标准偏差(RSD)均小于2.8%,峰电流的RSD均小于4.0%。将所建立的方法用于11种实际茶叶样品中Arg、Trp、Phe和Tyr含量的测定,结果令人满意。该方法可以为茶叶的质量评估提供借鉴。     

A cytochrome c variant resistant to heme degradation by hydrogen peroxide

BACKGROUND: Cytochrome c has peroxidase-like activity and can catalyze the oxidation of a variety of organic substrates, including aromatic, organosulfur and lipid compounds. Like peroxidases, cytochrome c is inactivated by hydrogen peroxide. During this inactivation the heme prosthetic group is destroyed. RESULTS: Variants of the iso-1-cytochrome c were constructed by site-directed mutagenesis and were found to be more stable in the presence of hydrogen peroxide than the wild type. No heme destruction was detected in a triple variant (Tyr67-->Phe/Asn52-->Ile/Cys102-->Thr) with the catalytic hydrogen peroxide concentration of 1 mM, even following the loss of catalytic activity, whereas both double variants Tyr67-->Phe/Cys102-->Thr and Asn52-->Ile/Cys102-->Thr showed a greater rate of peroxide-induced heme destruction than observed with the wild-type protein. CONCLUSIONS: Heme destruction and catalytic inactivation are two independent processes. An internal water molecule (Wat166) is shown to be important in the heme destruction process. The absence of a protein radical in the resistant variant suggests that the protein radical is necessary in the heme destruction process, but presumably is not involved in the reactions leading up to the protein inactivation.     

Stabilizing interactions between aromatic and basic side chains in alpha-helical peptides and proteins. Tyrosine effects on helix circular dichroism

Here we investigate the structures and energetics of interactions between aromatic (Phe or Tyr) and basic (Lys or Arg) amino acids in alpha-helices. Side chain interaction energies are measured using helical peptides, by quantifying their helicities with circular dichroism at 222 nm and interpreting the results with Lifson-Roig-based helix/coil theory. A difficulty in working with Tyr is that the aromatic ring perturbs the CD spectrum, giving an incorrect helicity. We calculated the effect of Tyr on the CD at 222 nm by deriving the intensities of the bands directly from the electronic and magnetic transition dipole moments through the rotational strengths corresponding to each excited state of the polypeptide. This gives an improved value of the helix preference of Tyr (from 0.48 to 0.35) and a correction to the helicity for the peptides containing Tyr. We find that Phe-Lys, Lys-Phe, Phe-Arg, Arg-Phe, and Tyr-Lys are all stabilizing by -0.10 to -0.18 kcal.mol-1 when placed i, i + 4 on the surface of a helix in aqueous solution, despite the great difference in polarity between these residues. Interactions between these side chains have previously been attributed to cation-pi bonds. A survey of protein structures shows that they are in fact predominantly hydrophobic interactions between the CH2 groups of Lys or Arg and the aromatic rings.     

Syntheses and biological activities of endothelin-1 analogs.

Endothelin-1 analogs replaced by various amino acids at position 21, namely [X21]-ET-1, were synthesized, and their agonistic vasoconstrictor activity on rat thoracic aortic strips and receptor binding activity on rat brain membrane fraction were examined to elucidate their structure-activity relationship. The vasoconstrictor activities of [Tyr21]- and [Phe21]-ET-1 were one order of magnitude smaller than that of ET-1, and those of [His21]-, [Gly21]-, [Ser21]-, [Ala21]- and [Lys21]-ET-1 were more than two orders of magnitude smaller than that of ET-1. On the other hand, the replacements by Ile, Glu, Gln and Pro resulted in distinguished losses of the vasoconstrictor activities. In addition, preincubation with these analogs did not blunt ET-1-induced vasoconstriction and showed no antagonistic activity. The binding inhibitory activities of these analogs against 125I-ET-1 were approximately conformable to the vasoconstrictor activities with only a slight exception. These findings demonstrate that the phenyl group at position 21 is important for both the vasoconstrictor activity and the receptor binding activity.     

Resonance Energy Transfer Relates the Gas‐Phase Structure and Pharmacological Activity of Opioid Peptides

Enkephalins are efficient pain‐relief drugs that bind to transmembrane opioid receptors. One key structural parameter that governs the pharmacological activity of these opioid peptides and is typically determined from condensed‐phase structures is the distance between the aromatic rings of their Tyr and Phe residues. We use resonance energy transfer, detected by a combination of cold ion spectroscopy and mass spectrometry, to estimate the Tyr–Phe spacing for enkephalins in the gas phase. In contrast to the condensed‐phase structures, these distances appear to differ substantially in enkephalins with different pharmacological efficiencies, suggesting that gas‐phase structures might be a better pharmacophoric metric for ligand peptides.     

Retention behavior of peptides in hydrophilic-interaction chromatography

Selected hydrophilic interaction chromatography (HILIC) columns packed with bare silica, bridge-ethyl hybrid silica, or an amide sorbent chemistry were utilized for an investigation of chromatographic behavior and separation selectivity of tryptic peptides. Retention model was proposed allowing for retention prediction of peptides with correlation coefficient R(2)~0.92-0.97 for various columns. The values of optimized amino acid retention coefficients were compared to those obtained for reversed-phase liquid chromatography (Gilar et al., Anal. Chem. 2010, 82, 265-275) and used to elucidate the impact of different amino acid on peptide HILIC retention. In contrast to reversed-phase chromatography, where presence of Phe, Trp, Ile, and Leu amino acid residues in sequence strongly promoted, and presence of hydrophilic His, Lys and Arg residues strongly reduced peptide retention, the effects of these amino acid residues in HILIC were opposite (His, Lys and Arg promote, Phe, Trp, Ile and Leu demote peptide retention in HILIC). Retention coefficient optimized for pH experiments illustrated the impact of silanols on HILIC retention.     

Solid phase synthesis and opioid receptor binding activities of [D-Ala2, D-Leu5]enkephalin analogs containing a fluorinated aromatic amino acid

Five [D-Ala2, D-Leu55]enkephalin (DADLE) analogs containing fluorinated Tyr1 or Phe4 residue, that is, [Phe(2F)4] (I), [Phe(3F)4] (II), [Phe(4F)4] (III), [Tyr(3F)1] (IV) and [Tyr(2F)1] (V), were synthesized by the solid phase method and their opioid receptor affinities were examined. Affinity profiles of five derivatives for the mu- and delta-receptor were similar to those of DADLE, and the affinity for kappa-receptor was zero or negligible.     

Electron capture dissociation product ion abundances at the X amino acid in RAAAA-X-AAAAK peptides correlate with amino acid polarity and radical stability

We present mechanistic studies aimed at improving the understanding of the product ion formation rules in electron capture dissociation (ECD) of peptides and proteins in Fourier transform ion cyclotron resonance mass spectrometry. In particular, we attempted to quantify the recently reported general correlation of ECD product ion abundance (PIA) with amino acid hydrophobicity. The results obtained on a series of model H-RAAAAXAAAAK-OH peptides confirm a direct correlation of ECD PIA with X amino acid hydrophobicity and polarity. The correlation factor (R) exceeds 0.9 for 12 amino acids (Ile, Val, His, Asn, Asp, Glu, Gln, Ser, Thr, Gly, Cys, and Ala). The deviation of ECD PIA for seven outliers (Pro is not taken into consideration) is explained by their specific radical stabilization properties (Phe, Trp, Tyr, Met, and Leu) and amino acid basicity (Lys, Arg). Phosphorylation of Ser, Thr, and Tyr decreases the efficiency of ECD around phosphorylated residues, as expected. The systematic arrangement of amino acids reported here indicates a possible route toward development of a predictive model for quantitative electron capture/transfer dissociation tandem mass spectrometry, with possible applications in proteomics.     

3D-QSAR and molecular mechanics study for the differences in the azole activity against yeastlike and filamentous fungi and their relation to P450DM inhibition. 1. 3-substituted-4(3H)-quinazolinones

A combination between 3D-QSAR and molecular mechanics (MM)-docking study was used as a tool to detail and model the mechanism of action of 46 antifungal azoles. Two methods of alignment of the ligands were performed: (i) alignment of the main skeleton without substituents and (ii) alignment of a defined substructure. The best model is characterized by q(2) with the values of 0.70 for yeastlike (yeast), 0.66 for filamentous fungi, and 0.70 for the selectivity against filamentous fungi. 3D-QSAR regression maps derived from six models were used to identify the regions responsible for the differences in the compounds activity against yeast and filamentous fungi. The binding energy of the important substructures (Local Binding Energy-LBE) and its standard deviation were calculated in order to demonstrate quantitatively the contribution of substituents reflecting the diversity of the antifungal activity. The comparisons of these results with the same regions of the contour maps indicated a good correspondence between the 3D-QSAR and MM (LBE) approaches allowing association between the maps and the participating residues in the active sites of P450DM of C. albicans and A. fumigatus. The pi-pi interactions of two or more aromatic groups of the ligands with Phe228 and Tyr132 prove to be most important for the differences in activity against C. albicans. In A. fumigatus there was a better occupation of the inner central I-spiral in the areas around the heme. For the activity against A. fumigatus the pi-pi interactions of aromatic groups of the compounds with Phe509, Phe228, and Tyr132 are significant for the activity.