Biomedical subjects

X Chen

Publications and source records attributed to X Chen.

At least 1,045 records · Page 58Linked to original sources

PubMed1997

Smad4 and FAST-1 in the assembly of activin-responsive factor.

Members of the TGF-beta superfamily of signalling molecules work by activating transmembrane receptors with phosphorylating activity (serine-threonine kinase receptors); these in turn phosphorylate and activate SMADs, a class of signal transducers. Activins are growth factors that act primarily through Smad2, possibly in partnership with Smad4, which forms heteromeric complexes with different ligand-specific SMADs after activation. In frog embryos, Smad2 participates in an activin-responsive factor (ARF), which then binds to a promoter element of the Mix.2 gene. The principal DNA-binding component of ARF is FAST-1, a transcription factor with a novel winged-helix structure. We now report that Smad4 is present in ARF, and that FAST-1, Smad4 and Smad2 co-immunoprecipitate in a ligand-regulated fashion. We have mapped the site of interaction between FAST-1 and Smad2/Smad4 to a novel carboxy-terminal domain of FAST-1, and find that overexpression of this domain specifically inhibits activin signalling. In a yeast two-hybrid assay, the FAST-1 carboxy terminus interacts with Smad2 but not Smad4. Deletion mutants of the FAST-1 carboxy terminus that still participate in ligand-regulated Smad2 binding no longer associated with Smad4 or ARF. These results indicate that Smad4 stabilizes a ligand-stimulated Smad2-FAST-1 complex as an active DNA-binding factor.

Activins↗

PubMed1997

Effects of prolonged hyperinsulinemia on serum leptin in normal human subjects.

We have studied the effect of prolonged hyperinsulinemia and hyperglycemia on serum leptin levels in young nonobese males during 72-h euglycemic-hyperinsulinemic and hyperglycemic ( approximately 8.5 and 12.6 mM) clamps. Hyperinsulinemia increased serum leptin concentrations (by RIA) dose-dependently. An increase in serum insulin concentration of > 200 pM for > 24 h was needed to significantly increase serum leptin. An increase of approximately 800 pM increased serum leptin by approximately 70% over 72 h. Changes in plasma glucose concentrations (from approximately 5.0 to approximately 12.6 mM) or changes in plasma FFA concentrations (from < 100 to > 1,000 microM) had no effect on serum leptin. Serum leptin concentrations changed with circadian rhythmicity. The cycle length was approximately 24 h, and the cycle amplitude (peak to trough) was approximately 50%. The circadian leptin cycles and the circadian cycles of total body insulin sensitivity (i.e., GIR, the glucose infusion rates needed to maintain euglycemia during hyperinsulinemic clamping) changed in a mirror image fashion. Moreover, GIR decreased between Days 2 and 3 (from 11.4+/-0.2 to 9. 8+/-0.2 mg/kg min, P< 0.05) when mean 24-h leptin levels reached a peak. In summary, we found (a) that 72 h of hyperinsulinemia increased serum leptin levels dose-dependently; (b) that hyperglycemia or high plasma FFA levels did not affect leptin release; (c) that leptin was released with circadian rhythmicity, and (d) that 24-h leptin cycles correlated inversely with 24-h cycles of insulin sensitivity. We speculate that the close positive correlation between body fat and leptin is mediated, at least in part, by insulin.

Adult↗

PubMed1997

Modulation of yeast F-actin structure by a mutation in the nucleotide-binding cleft.

Although the actin sequence is very highly conserved across evolution, tissue-specific expression of different isoforms in high eukaryotes suggests that different isoforms carry out different functions. However, little information exists about either the differences in filaments made from different actins or the effects on filament structure caused by the various mutations in actin that have been introduced to gain insight into actin function. Using electron microscopy and three-dimensional reconstruction, we have studied the differences in the filaments made by yeast and rabbit skeletal muscle actin, two proteins with 88% homologous sequences, and we have assessed the changes in filament structure caused by the introduction of the S14A mutation into yeast actin. Elimination of the S14 hydroxyl group, assumed to bind to the gamma-phosphate of actin-bound ATP, results in a 40 to 60-fold decrease in actin's affinity for ATP. We show that yeast actin displays less extensive contacts between the two long-pitch helical strands than does muscle actin, and displays the large cooperativity within filaments previously observed for muscle actin. Finally, we demonstrate that the S14A mutation narrows the cleft between the two lobes of the actin subunit and strengthens the inter-strand connections in F-actin.

Actins↗

PubMed1997

Protein half-lives of calmodulin and the plasma membrane Ca-ATPase in rat brain.

We report the half-lives for two proteins involved in the regulation of intracellular calcium in the brain: the plasma membrane Ca-ATPase and its regulatory protein, calmodulin. [14C]-labeled leucine was injected into seven month old adult Fischer 344 rats and the time-dependent appearance and loss of radioactivity was monitored in both the serum and proteins from the brains of rats sacrificed from 4 hours to 13 days after injection. Experimental data obtained for calmodulin and the plasma membrane Ca-ATPase are best described by theoretical curves accounting for leucine reutilization that assume apparent half-lives of 18 (+/-2) hours and 12 (+/-1) days, respectively.

Animals↗

PubMed1997

Thermophoresis of a Small Evaporating Particle in a High-Temperature Diatomic Gas

Kinetic-theory analytical results are presented concerning the effect of intense evaporation on the thermophoretic force acting on a spherical particle suspended in a high-temperature diatomic gas for the case of free-molecule regime. Molecule dissociation and atom recombination are included in the analysis. It has been shown that evaporation may substantially enhance the thermophoretic force acting on a particle, especially for the case of the particle materials with low evaporation latent heat and small molecular weight and at high gas temperatures. The values of the effective atomic and molecular thermal-accommodation factors do not affect the thermophoretic force acting on a nonevaporating particle, but they affect significantly the evaporation-added thermophoretic force. It has been shown that the recombination fraction of atoms at the particle surface does not influence the thermophoresis.

Journal Article↗

PubMed1997

Investigation of the structural basis for thermodynamic stabilities of tandem GU wobble pairs: NMR structures of (rGGAGUUCC)2 and (rGGAUGUCC)2.

The symmetric, tandem GU mismatch motifs, and , which only differ in the mismatch order, have an average difference in thermodynamic stability of 2 kcal/mol at 37 degrees C. Thermodynamic studies of duplexes containing these motifs indicate the effect is largely localized to the mismatches and adjacent base pairs. The three-dimensional structures of two representative duplexes, (rGGAGUUCC)2 and (rGGAUGUCC)2, were determined by two-dimensional NMR and a simulated annealing protocol. Local deviations are similar to other intrahelical GU mismatches with little effect on backbone torsion angles and a slight overtwisting between the base pair 5' of the G of the mismatch and the mismatch itself. Comparisons of the resulting stacking patterns along with electrostatic potential maps suggest that interactions between highly negative electrostatic regions between base pairs may play a role in the observed thermodynamic differences.

Base Composition↗

PubMed1997

Impaired generation of bone marrow B lymphocytes in mice deficient in C/EBPbeta.

CAAT/enhancer binding proteins (C/EBP) are a family of transcription factors that mediates adipocyte differentiation and the regulation of genes expressed in immune responses and inflammation, such as interleukin-6 (IL-6), IL-8, and granulocyte colony-stimulating factor (G-CSF). We investigated the role of C/EBPbeta (NF-IL6) in the generation of bone marrow B lymphocytes by taking advantage of C/EBPbeta-/- mice. We found that the expansion of bone marrow (BM) B lymphocytes was impaired in long-term lymphoid cultures from C/EBPbeta-/- mice. Consistent with this finding, the number of BM B cells was decreased in C/EBPbeta-/- mice. Both the levels of IL-7 gene expression and bioactive IL-7 from BM stromal cells were decreased in C/EBPbeta-/- mice. Furthermore, the proliferative responsiveness of BM B-cell precursors to IL-7 was also reduced as compared to wild-type mice, indicating that C/EBPbeta is required for the generation of BM B cells induced by IL-7. Accordingly, IL-7 stimulates the C/EBPbeta DNA-binding activity of normal BM pre-B lymphocytes as well as of 70Z/3 pre-B cells. These results point to C/EBPbeta as a critical signaling molecule in BM B lymphopoiesis.

Animals↗

PubMed1997

Detection of antigen-antibody binding events with the atomic force microscope.

An atomic force microscope (AFM) has been used to directly monitor specific interactions between antibodies and antigens employed in an immunoassay system. Results were achieved using AFM probes functionalized with ferritin, and monitoring the adhesive forces between the probe and anti-ferritin antibody-coated substrates. Analysis of the force distribution data suggests a quantization of the forces, with a period of 49 +/- 10 pN. This periodic force may be attributed to single unbinding events between individual antigen and antibody molecules. These results demonstrate that the AFM could be employed as an analytical tool to study the interactions between the molecules involved in biosensor systems. The potential of the technique to provide information relating to the manner in which the antibody molecule binds to its specific antigen is also discussed.

Animals↗

PubMed1997

Streptococcal preparation OK-432 is a potent inducer of IL-12 and a T helper cell 1 dominant state.

Streptococcal preparation OK-432 is a bacterial immunopotentiator extensively used in Japan for adjuvant cancer therapy. Using a C57BL/6 mouse model, OK-432 was found to induce multiple cytokines including the Th1 polarizing cytokine IL-12. Expression of IL-12 protein by murine splenocytes was restricted to macrophages and B cells and led to high levels of IFN-gamma production from both CD4+ and CD8+ T cells. Of the Th2 cytokines IL-4 and IL-10, only IL-10 protein was detected and originated primarily from the adherent cell population. Its expression was delayed relative to IL-12. A similar pattern of cytokine induction was observed from human PBMCs. OK-432-driven IFN-gamma production was inhibited by anti-IL-12 Ab, anti-IL-2 Ab, anti-TNF-alpha Ab, and anti-IL-2R alpha Ab, suggesting that IFN-gamma production from Th1 cells is induced by the cooperation action of these cytokines through the IL-2R alpha pathway. When compared with another widely used immunopotentiator bacillus Calmette-Guérin (BCG), OK-432 was a stronger IL-12 and IFN-gamma inducer. Furthermore, the mechanism of IFN-gamma induction by OK-432 differed from BCG in that coincident granulocyte-macrophage CSF and IL-1 expression played little to no role. These results suggest that OK-432 is a potent multicytokine inducer, specifically a strong inducer of IL-12, and that OK-432 may exert its antitumor effect by promoting a Th1-dominant state.

Adjuvants, Immunologic↗

PubMed1997

HIV-1 Tat induces the expression of the interleukin-6 (IL6) gene by binding to the IL6 leader RNA and by interacting with CAAT enhancer-binding protein beta (NF-IL6) transcription factors.

Human immunodeficiency virus type 1 (HIV-1) infection is associated with severe psoriasis, B cell lymphoma, and Kaposi's sarcoma. A deregulated production of interleukin-6 (IL6) has been implicated in the pathogenesis of these diseases. The molecular mechanisms underlying the abnormal IL6 secretion of HIV-1-infected cells may include transactivation of the IL6 gene by HIV-1. Here we report the molecular mechanisms of Tat activity on the expression of the IL6 gene. By using 5' deletion mutants of pIL6Pr-CAT and using IL6:HIV-1-LTR hybrid constructs where discrete regions of the IL6 promoter replaced the TAR sequence in HIV-1 LTR, we identified a short sequence of the 5'-untranslated region of the IL6 mRNA that is required for Tat to trans-activate the IL6 promoter. This sequence acquires a stem-loop structure and includes a UCU sequence that binds to Tat and is necessary for full trans-activation. In addition, we provide the evidence that Tat can function by enhancing the CAAT enhancer-binding protein (C/EBP) DNA binding activity and is able to complex with in vitro translated C/EBPbeta, which is a major mediator of IL6 promoter function. By using the yeast two-hybrid system and immunoprecipitation, we observed that the interaction of Tat with C/EBP proteins also occurred in vivo. The data are consistent with the possibility that Tat may function on heterologous genes by interacting with RNA structures possibly present in a large number of cellular and viral genes. In addition, Tat may function by protein-protein interactions, leading to the generation of heterodimers with specific transcription factors.

CCAAT-Enhancer-Binding Proteins↗

PubMed1997

RGD-containing peptides trigger apoptosis in glomerular mesangial cells of adult human kidneys.

Substantial evidence was given that different Arg-Gly-Asp (RGD)-containing peptides, linear Arg-Gly-Asp-Ser (RGDS) and cyclic Arg-Gly-Asp-Phe (RGDF), which are sequences present in fibronectin and vitronectin and which can bind alpha5beta1 and alpha(v)beta3 integrins, respectively, both induced apoptosis and expression of interleukin-1beta-converting enzyme (ICE) in cultured glomerular mesangial cells from adult human kidneys, with consistent apoptosis features appearing as nuclei condensation and fragmentation, internucleosomal DNA fragmentation, and DNA content decrease. These results indicated that both fibronectin and vitronectin may be important in the survival of human glomerular mesangial cells and that stable cyclic RGD(D)FV peptide may be a candidate to be used for regulating apoptosis in vivo.

Adult↗

PubMed1997

Construction of a 1-Mb restriction-mapped cosmid contig containing the candidate region for the familial Mediterranean fever locus (MEFV) on chromosome 16p 13.3.

In this paper we describe the assembly and restriction map of a 1.05-Mb cosmid contig spanning the candidate region for familial Mediterranean fever (FMF), a recessively inherited disorder of inflammation localized to 16p13.3. Using a combination of cosmid walking and screening for P1, PAC, BAC, and YAC clones, we have generated a contig of genomic clones spanning approximately 1050 kb that contains the FMF critical region. The map consists of 179 cosmid, 15 P1, 10 PAC, 3 BAC, and 17 YAC clones, anchored by 27 STS markers. Eight additional STSs have been developed from the approximately 700 kb immediately centromeric to this genomic region. Five of the 35 STSs are microsatellites that have not been previously reported. NotI and EcoRI mapping of the overlapping cosmids, hybridization of restriction fragments from cosmids to one another, and STS analyses have been used to validate the assembly of the contig. Our contig totally subsumes the 250-kb interval recently reported, by founder haplotype analysis, to contain the FMF gene. Thus, our high-resolution clone map provides an ideal resource for transcriptional mapping toward the eventual identification of this disease gene.

Base Sequence↗

PubMed1997

Characterization of early pulmonary hyperproliferation and tumor progression and their inhibition by black tea in a 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced lung tumorigenesis model with A/J mice.

The pathogenesis of pulmonary tumors induced by a tobacco carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and its inhibition by black tea have been characterized. Female A/J mice (6 weeks old) were treated with a single dose of NNK (103 mg/kg of body weight, i.p.) on day 0, and the cell proliferation index was measured by the incorporation of bromodeoxyuridine (BrdUrd) immunohistochemically. The number of BrdUrd-labeled cells increased in the bronchiolar epithelium from day 2 to day 14, with the highest proliferation rate observed on day 5. By day 35, the BrdUrd-labeling index returned to the level of the control group. Further examination of the day 35 samples revealed the presence of foci of hyperproliferative cells in the bronchiolar epithelium, particularly in the bronchiolalveolar regions. These proliferating bronchiolar epithelial cells (Clara cells) may be the initiated sites for pulmonary tumorigenesis. In this short-term model, administration of black tea polyphenols (0.3%) through the drinking water starting 24 h after NNK treatment significantly inhibited NNK-induced early bronchiolar cell proliferation on day 5. In long-term studies, adenomas were observed in 100% (15 of 15) of the mice at week 16, with 7.8 +/- 0.8 tumors per mouse. At week 52, a malignant tumor incidence of 80% (41 of 51 mice) and a malignant tumor multiplicity of 2.39 +/- 0.19 were observed. The growth patterns of the malignant tumors, which included solid, papillary, and mixed types, may be associated with the cellular origin of the tumor. The cell proliferation indices, as measured by proliferating cell nuclear antigen immunohistochemistry, were significantly higher in dysplasia within adenoma than in adenoma, and significantly higher in adenoma at week 52 than in adenoma at week 16. Administration of black tea, starting 16 weeks after a single dose of NNK, inhibited the progression of adenoma to adenocarcinoma as determined by both malignant tumor incidence and multiplicity. The cell proliferation rate in adenomas was also suppressed by black tea treatment. The present work demonstrates the antiproliferative activities of black tea and its polyphenols. Such activities, at the early and late stages of lung tumorigenesis, may be important for the cancer-chemopreventive activities of black tea.

Animals↗

PubMed1997

Amyloid-beta peptide-receptor for advanced glycation endproduct interaction elicits neuronal expression of macrophage-colony stimulating factor: a proinflammatory pathway in Alzheimer disease.

In Alzheimer disease (AD), neurons are thought to be subjected to the deleterious cytotoxic effects of activated microglia. We demonstrate that binding of amyloid-beta peptide (Abeta) to neuronal Receptor for Advanced Glycation Endproduct (RAGE), a cell surface receptor for Abeta, induces macrophage-colony stimulating factor (M-CSF) by an oxidant sensitive, nuclear factor kappaB-dependent pathway. AD brain shows increased neuronal expression of M-CSF in proximity to Abeta deposits, and in cerebrospinal fluid from AD patients there was approximately 5-fold increased M-CSF antigen (P < 0.01), compared with age-matched controls. M-CSF released by Abeta-stimulated neurons interacts with its cognate receptor, c-fms, on microglia, thereby triggering chemotaxis, cell proliferation, increased expression of the macrophage scavenger receptor and apolipoprotein E, and enhanced survival of microglia exposed to Abeta, consistent with pathologic findings in AD. These data delineate an inflammatory pathway triggered by engagement of Abeta on neuronal RAGE. We suggest that M-CSF, thus generated, contributes to the pathogenesis of AD, and that M-CSF in cerebrospinal fluid might provide a means for monitoring neuronal perturbation at an early stage in AD.

Alzheimer Disease↗

PubMed1997

Transcription factor AP2 is required for expression of the rat transforming growth factor-alpha gene.

DNase I footprinting of the rat TGF alpha promoter in the presence of crude cell nuclear extract revealed three sites of protein-DNA interaction (Fp-A, Fp-B, Fp-C) in the region from -222 to +73. Mutation of specific sites within the Fp-A and Fp-B regions reduced expression of a TGF alpha promoter-reporter gene (TGF alphaLUC) from 50-90% in transiently transfected CHO cells, indicating the importance of protein/DNA interactions at these sites. Since Fp-A contained a perfect AP2 consensus sequence (5'-GCCNNNGGC-3') as its center, we investigated the possibility that AP2 binding is important for TGF alpha promoter activity. A double-stranded oligonucleotide spanning Fp-A displayed a distinct mobility shift in the presence of nuclear extract that was inhibited by an excess of known functional AP2-binding sequence. Moreover, a similar mobility shift occurred in the presence of purified AP2 protein, and the further addition of AP2 antibody produced a supershifted complex. More refined DNase I footprinting of a smaller, oligonucleotide probe in the presence of purified AP2 protein revealed a protected region that included the putative AP2 binding site. Additionally, co-transfection of an AP2 expression vector increased TGF alphaLUC expression 25-fold in Drosophila Schneider cells. These various findings corroborate a role for AP2 in TGF alpha promoter activity. The Fp-B region contains a T5 motif that has been previously suggested to function as an atypical TATA box. An Fp-B oligonucleotide displayed a specific gel mobility shift in the presence of a TATA binding protein (TBP)-TFIIA complex, and the further addition of TBP antibody produced a supershift. These results confirm that protein binding within Fp-B is functionally important, and they also indicate that the T5 motif functions as a TBP binding site.

Animals↗

PubMed1997

Crystal structures of the side-by-side binding of distamycin to AT-containing DNA octamers d(ICITACIC) and d(ICATATIC).

To understand the recognition interactions between AT-containing alternating DNA and minor groove binding drugs, the crystal structures of the side-by-side binding of two distamycin molecules to the DNA octamers d(ICITACIC)2 and d(ICATATIC)2, referred to here as TA and ATAT, respectively, have been determined at 1.6 A and 2.2 A, respectively. Compared to the previous 2:1 all-IC d(ICICICIC)2-distamycin complex, the substitutions of the I x C base-pairs by the A x T base-pairs enable the interactions of the drug with its natural target to be studied. Both complexes assume side-by-side drug binding, isomorphous to the all IC counterpart in the tetragonal space group P4(1)22 (a = b = 28.03 A, c = 58.04 A and a = b = 27.86 A, c = 58.62 A, respectively). The ATAT complex also crystallized in a new polymorphic monoclinic space group C2 (a = 33.38 A, b = 25.33 A, c = 28.11 A and beta = 120.45 degrees) and was solved at 1.9 A resolution. The structures of the three double drug x DNA complexes are very similar, characterized by systematic hydrogen bonding and van der Waals interactions. Each drug hydrogen bonds with the bases of the proximal DNA strand only and stacks with the sugar moiety, while the side-by-side drugs themselves exhibit pyrrole ring-peptide stacking. The pyrrole-peptide interaction is crucial for the side-by-side binding mode of the distamycin/netropsin family of drugs. The purine-pyrimidine alternation is probably responsible for the striking alternation in the helical and backbone conformations. The structures are conserved between the pure IC complex and the AT substituted complexes but further details of the side-by-side binding to DNA are provided by the 1.6 A resolution structure of TA.

Antiviral Agents↗

PubMed1997

Mammalian Mg2+-independent phosphatidate phosphatase (PAP2) displays diacylglycerol pyrophosphate phosphatase activity.

Recent studies indicate that the metabolism of diacylglycerol pyrophosphate (DGPP) is involved in a novel lipid signaling pathway. DGPP phosphatases (DGPP phosphohydrolase) from Saccharomyces cerevisiae and Escherichia coli catalyze the dephosphorylation of DGPP to yield phosphatidate (PA) and then catalyze the dephosphorylation of PA to yield diacylglycerol. We demonstrated that the Mg2+-independent form of PA phosphatase (PA phosphohydrolase, PAP2) purified from rat liver catalyzed the dephosphorylation of DGPP. This reaction was Mg2+-independent, insensitive to inhibition by N-ethylmaleimide and bromoenol lactone, and inhibited by Mn2+ ions. PAP2 exhibited a high affinity for DGPP (Km = 0.04 mol %). The specificity constant (Vmax/Km) for DGPP was 1. 3-fold higher than that of PA. DGPP inhibited the ability of PAP2 to dephosphorylate PA, and PA inhibited the dephosphorylation of DGPP. Like rat liver PAP2, the Mg2+-independent PA phosphatase activity of DGPP phosphatase purified from S. cerevisiae was inhibited by lyso-PA, sphingosine 1-phosphate, and ceramide 1-phosphate. Mouse PAP2 showed homology to DGPP phosphatases from S. cerevisiae and E. coli, especially in localized regions that constitute a novel phosphatase sequence motif. Collectively, our work indicated that rat liver PAP2 is a member of a phosphatase family that includes DGPP phosphatases from S. cerevisiae and E. coli. We propose a model in which the phosphatase activities of rat liver PAP2 and the DGPP phosphatase of S. cerevisiae regulate the cellular levels of DGPP, PA, and diacylglycerol.

Amino Acid Sequence↗

PubMed1997

Phospholipase A2 engineering. Structural and functional roles of the highly conserved active site residue aspartate-99.

The aspartate-99 of secreted phospholipase A2 (PLA2) has been proposed to be critical for the catalytic mechanism and interfacial activation of PLA2. Aspartate-99 connects the catalytic machinery (including the catalytic diad, the putative catalytic waters W5 and W6, and the calcium cofactor) to the hydrogen-bonding network. The latter involves Y52, Y73, the structural water, and the N-terminal region putatively required for the interfacial activation. A triple mutant of bovine pancreatic PLA2 with substitutions aspartate plus adjacent tyrosine residues (Y52,73F/D99N) was constructed, its X-ray structure was determined, and kinetic characteristics were analyzed. The kinetic properties of the D99N mutant constructed previously were also further analyzed. The X-ray structure of the Y52,73F/D99N mutant indicated a substantial disruption of the hydrogen-bonding network including the loss of the structural water similar to that seen in the structure of the D99N mutant published previously [Kumar, A., Sekharudu, Y. C., Ramakrishnan, B., Dupureur, C. M., Zhu, H., Tsai, M.-D., & Sundaralingam, M. (1994) Protein Sci. 3, 2082-2088]. Kinetic analysis demonstrated that these mutants possessed considerable catalytic activity with a k(cat) value of about 5% compared to WT. The values of the interfacial Michaelis constant were also little perturbed (ca. 4-fold lower for D99N and marginally higher for Y52,73F/D99N). The results taken together suggest that the hydrogen-bonding network is not critically important for interfacial activation. Instead, it is the chemical step that is perturbed, though only modestly, in the mutants.

Amino Acid Sequence↗