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H Kohno

Publications and source records attributed to H Kohno.

At least 163 records · Page 9Linked to original sources
PubMed1996

Molecular characterization of Euglena ascorbate peroxidase using monoclonal antibody.

Ascorbate peroxidase (EC 1.11.1.11) has been purified to electrophoretic homogeneity from Euglena gracilis Z. The enzyme showed a molecular mass of 58 kDa on SDS-PAGE and gel filtration, indicating that Euglena ascorbate peroxidase exists as a monomeric form. The substrate specificity for an electron donor and the stability of the purified enzyme were similar to those of cytosolic isozymes from higher plants. One of the characteristic properties was that Euglena ascorbate peroxidase reduces organic hydroperoxides as well as hydrogen peroxide. The N-terminal amino-acid sequence showed no significant similarity to any other ascorbate peroxidase from higher plants. However, the sequence of the peptides from the purified enzyme exhibited a high degree of homology to sequences of cytosolic and chloroplastic ascorbate peroxidases. Monoclonal antibodies against the purified Euglena ascorbate peroxidase were prepared. Two monoclonal antibodies (EAP1 and EAP2) showed high homology to cytosolic ascorbate peroxidases of higher plants, as judged by Western blot analysis. The EAP1 was also specific for chloroplastic ascorbate peroxidase from spinach. These findings indicate that Euglena ascorbate peroxidase exists in highly homologous regions with the ascorbate peroxidases of higher plants.

Amino Acid Sequence↗
PubMed1996

Requirement of multiple DNA-protein interactions for inducible expression of RNR3 gene in Saccharomyces cerevisiae in response to DNA damage.

The RNR3 gene encodes the large subunit of ribonucleotide reductase. Transcription of this gene is induced 12-fold in response to DNA damage or by a DNA replication blocker. To investigate cis-acting regulation, deletion analysis of the promoter region of the RNR3 gene was performed and we identified two upstream-repressing sequences in the RNR3 regulatory region. An 18-base-pairs fragment, termed DNA-damage responsive element 1 (DRE1) located between -212 and -194 in this region was found to be essential for the induction of RNR3. This fragment contained a negatively acting sequence where a protein factor bound to the region during normal growth but disappeared by exposure to 4-nitroquinoline-1-oxide. The other repressive element homologue to DRE1 was located at -263 to -254. One possible upstream-activating sequence which regulates the basal expression of RNR3 was also found. These results show that at least three potential cis-elements are necessary for the inducible expression of yeast expression of yeast RNR3 in response to DNA damage.

4-Nitroquinoline-1-oxide↗
PubMed1996

Hepatocyte growth factor induces transglutaminase activity that negatively regulates the growth signal in primary cultured hepatocytes.

Transglutaminase (TGase) activity increased 2.5-fold at 6 h after treatment of rat hepatocytes with 117 nM hepatocyte growth factor (HGF). In the same manner, putrescine incorporation into proteins of cells occurred in HGF-treated cells but did not in those pretreated with monodansylcadaverine (MDC), a TGase inhibitor, even in the presence of HGF. These results suggest that HGF-induced TGase was active and catalyzed some cross-linkage reaction. Cycloheximide completely blocked the increase in TGase activity induced by HGF, suggesting that HGF stimulated de novo synthesis of TGase within 6 h. Both [35S]methionine incorporation and Northern blotting analyses supported this possibility. Pretreatment of cells with MDC additionally increased HGF-induced DNA synthesis and the ratio of cells in S-phase. Similarly, TGase antisense oligonucleotide inhibited de novo synthesis of TGase, resulting in increase in the ratio of S-phase cells in the presence of HGF. Analyses of cross-linking of HGF to the receptor indicated that the antisense oligonucleotide inhibited the downregulation of HGF receptor subsequent to HGF-addition. These results provide the first evidence for inducibility of de novo synthesis of TGase by HGF and suggest that TGase negatively regulates the growth signal of HGF through the downregulation of receptor.

Animals↗
PubMed1996

Mouse coproporphyrinogen oxidase is a copper-containing enzyme: expression in Escherichia coli and site-directed mutagenesis.

We previously isolated cDNA for mouse coproporphyrinogen oxidase (CPO) and provided evidence for the induction of mRNA during differentiation of murine erythroleukemia cells (Kohno et al. (1993) J. Biol. Chem. 268, 21359-21363). To better understand the structure and the mechanisms of reaction of the enzyme, we expressed mouse CPO in Escherichia. coli and purified it to a homogeneity. Analysis of the metal content revealed that the recombinant mouse CPO contains one copper atom per polypeptide chain. When the bacterial cells were treated with D-penicillamine, a copper chelator, formation of the active CPO was partially reduced. Addition of Cu2+ in minimal medium resulted in 6-fold higher level of CPO activity. These results suggest that expression of active mouse CPO in E. coli depended on the presence of Cu2+ in the culture medium. To elucidate the apparent involvement of Cu2+ in enzyme function, a series of mutant enzymes, whose highly conserved histidine and cysteine residues were individually converted to alanine residue, were prepared by site-directed mutagenesis. Mutant enzymes were expressed in E. coli and their activities examined. Mutation at histidine 158 resulted in a complete loss of enzyme activity, yet the enzyme protein was expressed at a comparable level. Concomitantly, only a trace amount of Cu2+ was detected in the purified H158A enzyme. We propose that mouse CPO is copper-containing enzyme and Cu2+ interacts with a conserved histidine residue.

Amino Acid Sequence↗
PubMed1996

Transglutaminase induced by epidermal growth factor negatively regulates the growth signal in primary cultured hepatocytes.

Transglutaminase (TGase) activity increased 2.5-fold at 6 h after treatment of rat hepatocytes with 17 nM epidermal growth factor (EGF). In the same manner, putrescine incorporation into the proteins of cells occurred in EGF-treated cells, but not in those pretreated with monodansylcadaverine (MDC), a TGase inhibitor, even in the presence of EGF. These results suggest that EGF-induced TGase was active and catalysed some cross-linkage reaction. Cycloheximide completely blocked the increase in TGase activity induced by EGF, suggesting that EGF stimulated de novo synthesis of TGase within 6 h. Furthermore, Northern blotting analysis indicated that EGF increased the expression of TGase mRNA. Pretreatment of cells with MDC additionally increased EGF-induced DNA-synthesis and the ratio of cells in S-phase. TGase antisense oligonucleotide inhibited de novo synthesis of TGase, resulting in increases in the ratios of S- and G2/M-phase cells in the presence of EGF. This effect was the result of inhibition of EGF-induced down-regulation of high-affinity receptor expression. These results suggest that the EGF-induced increase in TGase activity is a negative regulator of a growth signal in rat hepatocytes.

Animals↗
PubMed1996

Repeat hepatectomy for recurrent hepatocellular carcinoma.

This study evaluated the results of repeat hepatectomy for recurrent hepatocellular carcinoma (HCC) and delineated the limitations of such a treatment strategy. Of 290 patients who had radical hepatic resection, 167 had intrahepatic tumour recurrence. Fifty patients underwent a second hepatectomy. Third and fourth hepatectomies were carried out on six and two patients respectively. There were 36 men and 14 women. Age ranged from 32 to 77 years (mean 59.1 years). Liver cirrhosis was present in 38 and chronic hepatitis in 12 patients. Operative death occurred in four patients (8 per cent) after a second and in one of six after a third hepatectomy, compared with 4.5 per cent after first hepatectomy. The five postoperative deaths were attributed to either Child grade B or C liver disease, or severe intraoperative bleeding. The 5-year survival rate was better in these 50 patients (41 per cent) than in 117 patients who were treated otherwise (7 per cent). The present study indicates that repeat hepatectomy may be useful for recurrent HCC. It is indicated principally for patients with a solitary tumour and Child grade A liver disease.

Adult↗
PubMed1996

1 alpha,25-dihydroxyvitamin D3 inhibits cell growth and chondrogenesis of a clonal mouse EC cell line, ATDC5.

Here we report the effects of 1 alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3] in vitro on the growth and chondrogenesis of a chondroprogenitor-like clonal mouse EC cell line, 10(-10) to 10(-7) M ATDC5. 1,25(OH)2D3 inhibited [3H]thymidine incorporation in undifferentiated chondroprogenitor-like ATDC5 cells in time- and dose-dependent manners. 1,25(OH)2D3 suppressed cartilage-nodule formation and the accumulation of cartilage-specific proteoglycan in ATDC5 cells in a dose-dependent manner. The 1,25(OH)2D3-induced inhibition of cartilage-nodule formation was reversible and direct, unrelated to the antiproliferative action of the hormone on the undifferentiated ATDC5 cells. ATDC5 cells even in the precartilaginous stage expressed 4.4 kb vitamin D receptor (VDR) mRNA as assessed by northern blot analysis. The equilibrium saturation binding experiment revealed the presence of a single class of saturable and high-affinity binding sites for 1,25(OH)2D3 in the cytosols. These results provide evidence for the hypothesis that both recruitment and chondrogenesis of chondroprogenitors are negatively regulated by 1,25(OH)2D3 via a VDR-mediated process in vivo.

Animals↗
PubMed1996

Estimation of coronary flow reserve with the instantaneous coronary flow velocity versus pressure relation: a new index of coronary flow reserve independent of perfusion pressure.

Our goal was to develop a new method to evaluate coronary flow reserve independent of perfusion pressure by equipping dogs with a Doppler flow probe in the left anterior descending artery. The slope of the linear portion of the velocity-pressure loop in the late diastolic phase was defined as VP slope; the hyperemic:basal ratio of the VP slope was defined as VP slope ratio, and the hyperemic:basal ratio of the mean coronary blood flow velocity was defined as coronary flow reserve (CFR). We measured VP slope ratio and CFR, altering aortic pressure by aortic banding and inferior vena cava occlusion. VP slope ratio was independent of aortic pressure, but CFR increased with increments in aortic pressure. The VP slope ratio and CFR decreased in the presence of coronary stenoses. In conclusion, VP slope ratio is considered to be a physiologic index of the severity of coronary stenosis, which is independent of perfusion pressure. This measure is easily applicable in clinical practice.

Angioplasty, Balloon, Coronary↗
PubMed1996

Inhibition of growth and induction of TGF-beta 1 in human hepatocellular carcinoma with androgen receptor by cyproterone acetate in male nude mice.

BACKGROUND/AIMS: Hepatocellular carcinoma possesses androgen receptor but its true role is not known. This study aimed to investigate the effect of an anti-androgen cyproterone acetate on the growth of androgen receptor-positive hepatocellular carcinoma. METHODS: Androgen receptor-positive human hepatocellular carcinoma cells (KYN-1/SM-10) were subcutaneously transplanted into male nude mice. When the tumor size was about 10 mm, animals were subcutaneously administered cyproterone acetate (0.1 mg/day and 0.8 mg/day) or solvent alone for 21 days. Animals were serially sacrificed for measurements of testicular weight, tumor size, and cytosolic and nuclear androgen receptor levels in tumor. Proliferating cell nuclear antigen, transforming growth factor-alpha, and transforming growth factor-beta 1 in tumor were investigated immunohistochemically, using monoclonal antibodies. Apoptotic activity was also studied by the in situ DNA nick end labeling method. RESULTS: Cyproterone acetate depressed testicular weight, suppressed tumor growth, and decreased both cytosolic-androgen receptor and nuclear-androgen receptor levels dose-dependently. Numbers of proliferating cell nuclear antigen-positive cells were decreased transiently with the low dose but continuously with the high dose of cyproterone acetate. Transforming growth factor-alpha expression was not influenced by cyproterone acetate, but the high dose of cyproterone acetate induced higher expression of transforming growth factor-beta 1, associated with increased numbers of apoptotic tumor cells, peaking on day 3. CONCLUSIONS: The inhibition of growth of androgen receptor-positive hepatocellular carcinoma with cyproterone acetate in male nude mice could be due to G1-phase cell cycle arrest, and to some extent apoptosis induced by increased synthesis of transforming growth factor-beta 1 in tumor, caused by the direct action of cyproterone acetate through androgen receptors, as well as decreased testosterone levels in blood due to cyproterone acetate-induced testicular atrophy.

Androgen Antagonists↗
PubMed1996

Mutations in the envelope gene of hepatitis B virus variants co-occurring with antibody to surface antigen in sera from patients with chronic hepatitis B.

Three clones of hepatitis B virus (HBV) DNA were propagated from sera of each of five patients with chronic hepatitis B who possessed hepatitis B surface antigen (HBsAg) and antibody to HBsAg in their serum. The clones were sequenced within the envelope gene (the preS1, preS2 regions and the S gene). Clones from four patients had various missense mutations involving codons 124-147 of the S-gene which encode amino acids in the loop structures that form the conformational, common antigenic determinant of HBsAg. Clones from three patients had Asn-130 (Gly in the wild-type), which generated a potential N-glycosylation site, Asn-Thr-Ser, spanning amino acids 130-132 of the S-gene product. In addition, clones from one patient had Arg-145 (Gly in the wild-type), which has been reported in escape mutants of HBV. One of the three clones from another patient had Ser-126 in place of lle or Thr in wild-type HBV, but the remaining two had no mutations known to affect expression of the common determinant of HBsAg. The remaining patient possessed HBsAg of subtype adr and anti-HBs specific for the w determinant. Clones from this patient did not reveal any mutations which are known to affect the common antigenic determinant of HBsAg.

Amino Acid Sequence↗
PubMed1996

Effectiveness and limitations of the fallback I algorithm for transient supraventricular tachyarrhythmias in DDD pacing.

To examine the function of the fallback I algorithm (Chorus I, Ela Medical Inc., Montrouge, France), which automatically changes the DDD mode to VDI during transient supraventricular tachyarrhythmias to prevent high-rate tracking of the venticule, a total of 45 patients who were preoperatively diagnosed with sick-sinus syndrome (SSS) (Group 1; n = 19) or with advanced or complete atrioventricular block (AVB) (Group 2; n = 26) were followed up and analyzed. Mean follow-up times (mean +/- SD) were 22.4 +/- 9.7 and 12.4 +/- 10.9 months, respectively. Each of the groups was further divided into subgroups according to the preoperative existence of of paroxysmal atrial fibrillation (PAF) or paroxysmal supraventricular tachyarrhythmia (PSVT). During follow-up, the fallback started through a given cycle of ventricular pacing at a upper-rate limit (URL) to avoid a continuous high-rate tracking during the arrhythmias specifically in the patients with PSVT (0/4) (p = 0.0004). The fallback, however, sometimes started in the patients who had AVB not associated with either PAF or PSVT (4/21) during normal exercise because the fallback algorithm did not distinguish a normal P wave from the abnormal atrial waves. To further clarify the behavior of the fallback, a treadmill test was conducted in 25 of the 45 patients. The fallback start was observed in 12 of the 17 patients with AVB (Group 2). In such patients, the use of a long fallback delay and/or a high URL setting prevented the fallback starts during normal exercise. These results suggest that, in DDD pacing, the algorithm in Chorus I is useful in patients with SSS or AVB to avoid highrate tracking of the ventricle during transient supraventricular tachyarrhythmias, but special care must be taken to avoid the fallback starts during the normal exercise in AVB patients.

Adult↗
PubMed1996

Use of automatic mode change between DDD and AAI to facilitate native atrioventricular conduction in patients with sick sinus syndrome or transient atrioventricular block.

The benefits of the automatic DDD (DDD/AMC) mode in the Chorus II pacemaker (Chorus 6234; Ela Medical Inc.), which automatically switches the modes between DDD and AAI to respect spontaneous AV conduction as much as possible in AAI while preserving safety pacing in DDD during paroxysmal AV block (AVB) only, remain unproven. This study examined the functions of the DDD/AMC mode in 12 patients with sick sinus syndrome (SSS; n = 10) or paroxysmal complete AVB (n = 2). A short-term (24 hours) comparative study between simple DDD mode and the DDD/AMC mode was performed in 8 of the 12 patients, and a medium-term (55.2 +/- 54.6 days) follow-up of the DDD/AMC mode was completed in all 12 patients. A comparative pair of 24-hour surface Holter ECGs was obtained in 6 of the 8 patients in the short-term study. Telemetry and built-in Holter histograms were collected in the outpatient clinic in all 12 patients. Although the percentage atrial pacing of the telemetry increased from 59.2 +/- 35.4 in DDD to 70.4 +/- 31.8 in DDD/AMC (P < 0.009; n = 8), the percentage ventricular pacing decreased from 64.6 +/- 37.7 in DDD to 36.2 +/- 43.1 in DDD/AMC (P < 0.027) in the short-term study. In particular, the reduction of percentage ventricular pacing to < 10% was observed in four patients with SSS not associated with > or = first-degree (1 degree) AVB on preoperative ECGs. Between the two modes a significant difference in arrhythmic events was not observed by the 24-hour surface Holter ECGS taken from the six patients in the short-term study. AAI-DDD switching associated with automatic modulation of AV delay and AV hysteresis occurred in all patients in the medium-term study. From the medium-term study, the total AV delay (AV delay plus AV hysteresis) exceeded 300 ms in 6 of the 12 patients in DDD/AMC, and usually became longest during nighttime. From the short- or medium-term study in the 12 patients, two patients preferred the DDD/AMC mode while one preferred the DDD mode. These results suggest that the DDD/AMC mode is useful, at least in SSS patients without > or = 1 degree AVB, by reducing the percentage ventricular pacing.

Adult↗
PubMed1996

Body composition, atherogenic risk factors and apolipoproteins following growth hormone treatment.

We studied the change in atherogenic risk factors in 27 children, 21 boys and 6 girls, 6 to 14 years of age, with growth hormone deficiency during 12 months of growth hormone replacement therapy. Changes in body composition and lipid profile during growth hormone treatment were evaluated. The atherogenic index was calculated using the equation [(total cholesterol- high-density lipoprotein cholesterol)(apolipoprotein B)]/[(apolipoprotein AI)(high-density lipoprotein cholesterol)]. Body fat decreased (p < 0.01), associated with an increase in lean body mass (p < 0.01). Total cholesterol and high-density lipoprotein cholesterol showed no significant changes. The atherogenic index significantly decreased from 1.44 +/- 0.60 to 1.09 +/- 0.52 (p < 0.01) after 12 months. Apolipoproteins CII and CIII increased throughout the study period (p < 0.01). Lipoprotein(a) and apolipoproteins AI, B and B/AI ratio did not change significantly. In conclusion, growth hormone treatment improved body composition and reduced atherogenic risk factors in children with growth hormone deficiency.

Adolescent↗
PubMed1996

ROM7/BEM4 encodes a novel protein that interacts with the Rho1p small GTP-binding protein in Saccharomyces cerevisiae.

The RHO1 gene encodes a homolog of the mammalian RhoA small GTP-binding protein in the yeast Saccharomyces cerevisiae. Rho1p is localized at the growth site and is required for bud formation. The RHO1(G22S, D125N) mutation is a temperature-sensitive and dominant negative mutation of RHO1, and a multicopy suppressor of RHO1(G22S, D125N), ROM7, was isolated. Nucleotide sequencing of ROM7 revealed that it is identical to the BEM4 gene (GenBank accession number L27816), although its physiological function has not yet been reported. Disruption of BEM4 resulted in the cold- and temperature-sensitive growth phenotypes, and cells of the deltabem4 mutant showed abnormal morphology, suggesting that BEM4 is involved in the budding process. The temperature-sensitive growth phenotype was suppressed by overexpression of RHO1, ROM2, which encodes a Rho1p-specific GDP/GTP exchange factor, or PKC1, which encodes a target of Rho1p. Moreover, glucan synthase activity, which is activated by Rho1p, was significantly reduced in the deltabem4 mutant. Two-hybrid and biochemical experiments revealed that Bem4p directly interacts with the nucleotide-free form of Rho1p and, to lesser extents, with the GDP- and GTP-bound forms of Rho1p, although Bem4p showed neither GDP/GTP exchange factor, GDP dissociation inhibitor, nor GTPase-activating protein activity toward Rho1p. These results indicate that Bem4p is a novel protein directly interacting with Rho1p and is involved in the RHO1-mediated signaling pathway.

Base Sequence↗
PubMed1996

Parathyroid hormone-related peptide delays terminal differentiation of chondrocytes during endochondral bone development.

To test the hypothesis that PTH-related peptide (PTHrP) is a paracrine regulator of endochondral bone development, we localized PTHrP and its cognate receptor during normal skeletal development at both messenger RNA (mRNA) and protein levels and compared the growth plate phenotypes of PTHrP-deficient [(PTHrP(-/-)] mice to those of normal littermates [PTHrP(+/+]. PTHrP mRNA was expressed adjacent to uncavitated joints, in the perichondrium of long bones and to a lower level in proliferating chondrocytes. In contrast, PTHrP protein was most evident at the interface of proliferating and hypertrophic zones, where it colocalized with PTH/PTHrP receptor mRNA and protein. Most strikingly, the proliferating zone was dramatically shorter in PTHrP(-/-) cartilage, although the percentage of cells in S-phase of the cell cycle in the proliferating zone was indistinguishable between PTHrP(+/+) and PTHrP(-/-) mice. Terminal differentiation of chondrocytes, which was characterized by cell hypertrophy, apoptosis (DNA fragmentation and decreased bcl-2 mRNA expression), and matrix mineralization, was more advanced in growth cartilage of PTHrP(-/-), compared with PTHrP(+/+) animals. These data demonstrate that PTHrP acts principally as a paracrine factor, which promotes elongation of endochondral bone by restraining or delaying the pace of chondrocytic development and terminal differentiation of growth-plate chondrocytes.

Animals↗
PubMed1996

Mutational analysis of the estrogen receptor ligand-binding domain: influence of ligand structure and stereochemistry on transactivation.

The mouse estrogen receptor (mER) exhibits ligand stereochemical specificity for indenestrol A (IA), a stilbestrol estrogen. IA has a chiral C3 methyl group, and the mER preferentially binds the S-enantiomer (IA-S), resulting in elevated biological activity when compared with the IA-R enantiomer. To elucidate the mechanisms for this stereochemical recognition, we have constructed a series of mERs with individual amino acid substitutions at Met521, His528, Met532, and Val537. The abilities of yeast-expressed wild-type and mutant mERs to transactivate an estrogen-responsive reporter gene construct were measured in the presence of diethylstilbestrol (DES) and IA enantiomers. The concentration of IA-S required to induce half-maximal transactivation by wild-type mER was 10-fold lower than IA-R, which is attributed to the 15-fold greater binding affinity for IA-S. Wild-type mER displayed similar dose-response curves for IA-R and demethyl IA, which lacks a C3 methyl group, demonstrating that the presence and correct orientation of the C3 methyl group on the IA compound is required for high-affinity ligand binding and transcriptional activity. Each mutant exhibited a reduced preference for IA-S enantiomer with respect to transactivation, suggesting that this region of the mER functions in ligand stereochemical recognition and activation. A mutation at Met532 diminished DES- and IA-S-induced transactivation by 7.5-fold and 40-fold respectively, with minimal change on their binding affinity. These data suggest that Met532 is required for transactivation induced by the potent agonist, IA-S, and the M532G mutation effectively uncouples IA-S ligand binding from transactivation. Use of these stereochemically different ligands in combination with mutagenesis of the mER demonstrates that ligand structure could influence transactivation by specifically altering the conformation of the mER AF-2 region.

Amino Acid Sequence↗
PubMed1995

A downstream target of RHO1 small GTP-binding protein is PKC1, a homolog of protein kinase C, which leads to activation of the MAP kinase cascade in Saccharomyces cerevisiae.

The RHO1 gene in Saccharomyces cerevisiae encodes a homolog of the mammalian RhoA small GTP-binding protein, which is implicated in various actin cytoskeleton-dependent cell functions. In yeast, Rho1p is involved in bud formation. A yeast strain in which RHO1 is replaced with RhoA shows a recessive temperature-sensitive growth phenotype. A dominant suppressor mutant was isolated from this strain. Molecular cloning of the suppressor gene revealed that the mutation occurred at the pseuodosubstrate site of PKC1, a yeast homolog of mammalian protein kinase C. Two-hybrid analysis demonstrated that GTP-Rho1p, but not GDP-Rho1p, interacted with the region of Pkc1p containing the pseudosubstrate site and the C1 domain. MKK1 and MPK1 encode MAP kinase kinase and MAP kinase homologs, respectively, and function downstream of PKC1. A dominant active MKK1-6 mutation or overexpression of MPK1 suppressed the temperature sensitivity of the RhoA mutant. The dominant activating mutation of PKC1 suppressed the temperature sensitivity of the RhoA mutant. The dominant activating mutation of PKC1 suppressed the temperature sensitivity of two effector mutants of RHO1, rho1(F44Y) and rho1(E451), but not that of rho1(V43T). These results indicate that there are at least two signaling pathways regulated by Rho1p and that one of the downstream targets is Pkc1p, leading to the activation of the MAP kinase cascade.

Amino Acid Sequence↗