Biomedical subjects

W Yan

Publications and source records attributed to W Yan.

At least 109 records · Page 6Linked to original sources

PubMed1998

Chromosome 22q11.2 interstitial deletions among childhood-onset schizophrenics and "multidimensionally impaired".

Since its first description almost a century ago schizophrenia with childhood onset, a rare yet devastating disorder, has been diagnosed in children as young as age 5. Recently, the velocardiofacial syndrome, whose underlying cause is interstitial deletions of 22q11.2, was found in 2 of 100 cases of schizophrenics with adult onset [Karayiorgou et al., Proc Natl Acad Sci USA 92: 7612-7616, 1995]. No study has documented the prevalence of velocardiofacial syndrome and the 22q11.2 deletion in a population of schizophrenics with childhood onset. Here we describe the result of such a study in a sample originally selected for a trial of atypical antipsychotic drugs. A separate group of patients was also included in the study; they can best be accounted for as a variant of childhood-onset schizophrenia (COS) and had been provisionally termed "multidimensionally impaired." Fluorescent in situ hybridization screening of 32 COS and 21 multidimensionally impaired patients revealed 1 COS patient with an interstitial deletion spanning at least 2.5 megabases.

Child↗

PubMed1998

The hydrophobic domains in the carboxyl-terminal signal for GPI modification and in the amino-terminal leader peptide have similar structural requirements.

Proteins having a glycosyl-phosphatidylinositol (GPI) membrane anchor are synthesized with a carboxyl-terminal signal that is cleaved in the endoplasmic reticulum prior to GPI modification. The signal is characterized by a moderately hydrophobic domain downstream from the cleavage/modification site. The essential features of this domain were characterized using a truncated version of folate receptor (FR) type beta (FR-beta delta 5) in which its five carboxyl-terminal amino acid residues were deleted without affecting the efficiency of GPI modification. The amino acids at various positions in the hydrophobic domain were systematically altered and the extent of GPI modification of the recombinant proteins was determined by measuring [3H]folic acid binding at the cell surface, by Western blot analysis and from the sensitivity of the proteins to phosphatidylinositol-specific phospholipase C (PI-PLC). The results indicate that a threshold level of hydrophobicity exists at a single position below which the efficiency of GPI modification decreases with increasing hydrophilicity. Further, the hydrophobic domain is characterized by a hydrophobicity profile and not merely a minimum overall hydrophobicity. Thus, a leucine-rich core hydrophobic segment of six to eight amino acid residues is more sensitive to relatively small hydrophilic substitutions compared to its flanking regions and such mutations could be compensated by a hydrophobic substitution elsewhere within this core segment. Such a hydrophobicity profile is characteristic of the amino-terminal leader peptide. When the entire hydrophobic domain of the leader peptide of FR-beta (12 amino acid residues) was substituted with the hydrophobic domain of the GPI signal (13 amino acids), it was possible to obtain expression of FR-beta on the cell surface. In this construct, point mutations in the core hydrophobic segment and in the flanking regions within the substituting peptide produced a similar pattern of effects on the cell surface receptor expression compared to the corresponding mutations in the GPI signal of FR-beta. The results suggest that common principles may govern interactions of the hydrophobic domains of the GPI signal and the leader peptide with the endoplasmic reticulum.

Amino Acid Sequence↗

PubMed1998

Corticofugal modulation of the midbrain frequency map in the bat auditory system.

The auditory system, like the visual and somatosensory systems, contains topographic maps in its central neural pathways. These maps can be modified by sensory deprivation, injury and experience in both young and adult animals. Such plasticity has been explained by changes in the divergent and convergent projections of the ascending sensory system. Another possibility, however, is that plasticity may be mediated by descending corticofugal connections. We have investigated the role of descending connections from the cortex to the inferior colliculus of the big brown bat. Electrical stimulation of the auditory cortex causes a downward shift in the preferred frequencies of collicular neurons toward that of the stimulated cortical neurons. This results in a change in the frequency map within the colliculus. Moreover, similar changes can be induced by repeated bursts of sound at moderate intensities. Thus, one role of the mammalian corticofugal system may be to modify subcortical sensory maps in response to sensory experience.

Animals↗

PubMed1998

Protein kinase C beta regulates heterologous desensitization of thrombin receptor (PAR-1) in endothelial cells.

We studied the effects of protein kinase C (PKC) activation on endothelial cell surface expression and function of the proteolytically activated thrombin receptor 1 (PAR-1). Cell surface PAR-1 expression was assessed by immunofluorescence (using anti-PAR-1 monoclonal antibody), and receptor activation was assessed by measuring increases in cytosolic Ca2+ concentration in human dermal microvascular endothelial cells (HMEC) exposed to alpha-thrombin or phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA). Immunofluorescence showed that thrombin and TPA reduced the cell surface expression of PAR-1. Prior exposure of HMEC to thrombin for 5 min desensitized the cells to thrombin, indicating homologous PAR-1 desensitization. In contrast, prior activation of PKC with TPA produced desensitization to thrombin and histamine, indicating heterologous PAR-1 desensitization. Treatment of cells with staurosporine, a PKC inhibitor, fully prevented heterologous desensitization, whereas thrombin-induced homologous desensitization persisted. Depletion of PKC beta isozymes (PKC beta I and PKC beta II) by transducing cells with antisense cDNA of PKC beta I prevented the TPA-induced decrease in cell surface PAR-1 expression and restored approximately 60% of the cytosolic Ca2+ signal in response to thrombin. In contrast, depletion of PKC beta isozymes did not affect the loss of cell surface PAR-1 and induction of homologous PAR-1 desensitization by thrombin. Therefore, homologous PAR-1 desensitization by thrombin occurs independently of PKC beta isozymes, whereas the PKC beta-activated pathway is important in signaling heterologous PAR-1 desensitization in endothelial cells.

Calcium↗

PubMed1998

Effects of parathyroid hormone (PTH) and PTH-related peptide on expressions of matrix metalloproteinase-2, -3, and -9 in growth plate chondrocyte cultures.

The roles of PTH and PTH-related peptide (PTH-rp) in the expression of matrix metalloproteinases (MMPs) during endochondral bone formation were investigated, using various cartilages obtained from young rabbits and rabbit chondrocyte cultures. Immunohistochemical, immunoblotting, zymographical, and/or Northern blot analyses showed that MMP-2 and -9 levels were much higher in the growth plate than in permanent cartilage in vivo. In growth plate chondrocyte cultures, PTH, PTH-rp, and (Bu)2cAMP increased the amount of MMP-2 present in the culture medium, as revealed by zymograms and immunoblots, whereas the other tested growth factors or cytokines, including bone morphogenetic protein-2 and interleukin-1, did not increase the MMP-2 level. PTH also increased the MMP-2 messenger RNA level within 24 h. In addition, PTH increased MMP-3 and -9 levels in the growth plate chondrocyte cultures. However, in articular chondrocyte cultures, PTH had little effect on the levels of MMP-2, -3, and -9. In contrast to PTH, interleukin-1 induced MMP-3 and -9, but not MMP-2, in growth plate and articular chondrocytes. These findings suggest that in ossifying cartilage, PTH/PTH-rp plays a pivotal role in the induction of various MMPs, including MMP-2 (which is considered to be a constitutive enzyme), and that PTH/PTH-rp is involved in the control of cartilage-matrix degradation during endochondral bone formation.

Animals↗

PubMed1998

Vertebrate tinman homologues XNkx2-3 and XNkx2-5 are required for heart formation in a functionally redundant manner.

Tinman is a Drosophila homeodomain protein that is required for formation of both visceral and cardiac mesoderm, including formation of the dorsal vessel, a heart-like organ. Although several vertebrate tinman homologues have been characterized, their requirement in earliest stages of heart formation has been an open question, perhaps complicated by potential functional redundancy of tinman homologues. We have utilized a novel approach to investigate functional redundancy within a gene family, by coinjecting DNA encoding dominantly acting repressor derivatives specific for each family member into developing Xenopus embryos. Our results provide the first evidence that vertebrate tinman homologues are required for earliest stages of heart formation, and that they are required in a functionally redundant manner. Coinjection of dominant repressor constructs for both XNkx2-3 and XNkx2-5 is synergistic, resulting in a much higher frequency of mutant phenotypes than that obtained with injection of either dominant repressor construct alone. Rescue of mutant phenotypes can be effected by coinjection of either wild-type tinman homologue. The most extreme mutant phenotype is a complete absence of expression of XNkx2-5 in cardiogenic mesoderm, an absence of markers of differentiated myocardium, and absence of morphologically distinguishable heart on the EnNkxHD-injected side of the embryo. This phenotype represents the most severe cardiac phenotype of any vertebrate mutant yet described, and underscores the importance of the tinman family for heart development. These results provide the first in vivo evidence that XNkx2-3 and XNkx2-5 are required as transcriptional activators for the earliest stages of heart formation. Furthermore, our results suggest an intriguing mechanism by which functional redundancy operates within a gene family during development. Our experiments have been performed utilizing a recently developed transgenic strategy, and attest to the efficacy of this strategy for enabling transgene expression in limited cell populations within the developing Xenopus embryo.

Amino Acid Sequence↗

PubMed1998

[Analysis of MDMA and its metabolites in urine by GC and GC/MS].

The purpose of this study is to investigate the metabolism of MDMA in the person and establish the methods for the determination of MDMA and its metabolites in urine. MDMA and its metabolite were isolated from urine by liquid-liquid extraction after acidic or enzymatic hydrolysis and were determined by GC/MS(EI, PCI) and GC/FID. The results showed that MDMA was metabolized to MDA and HMMA in man. MDA in the urine after MDMA administration was approximately 0.10-0.14 that of the parent drug. The sensitivity limits of methods were 2-50 ng/ml, and the recoveries were greater than 85%(CV < 10%). It is concluded that the methods were simple, quick and accurate for determining urine concentration of MDMA addicts. MDA/MDMA Ratio can be used to indicate whether the parent drug is taken alone or in combination with its active metabolite.

3,4-Methylenedioxyamphetamine↗

PubMed1998

[Studies on flavones of Epimedium bevicorum Maxim].

OBJECTIVE: To systematically develop Epimedium spp. and control their quality. METHOD: Using polyamide and macroporous adsorbent resins AB-8. The compounds were identified on the basis of spectrometric data and physical and chemical test. RESULT: Four flavones were isolated from the herb. They were identified as wushanicariin, baohuoside VI, kaempferol-3,7-O-alpha-L-dirhamnoside and hexandraside E. CONCLUSION: These flavones were isolated from the herb and identified for the first time.

Drugs, Chinese Herbal↗

PubMed1998

Selective degradation of nonsense beta-phosphodiesterase mRNA in the heterozygous rd mouse.

PURPOSE: To investigate the molecular mechanism relating phenotype and genotype in the rd mouse, mRNA and pre-mRNA levels derived from the wild-type and position-347 nonsense mutant beta-phosphodiesterase (beta-PDE) genes were determined and compared with the corresponding gene copy ratios. METHODS: Total RNA and genomic DNA was isolated from the retinas of three heterozygous rd/+ mouse strains. For each, quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to determine the ratio of wild-type and rd beta-phosphodiesterase pre-mRNA and mature mRNA. The gene copy ratio between wild-type and rd beta-PDE was also determined by quantitative PCR. RESULTS: The pre-mRNA ratio of wild-type versus nonsense mutant was close to 1:1, whereas the corresponding mRNA ratio was greater than 3:1, even though the gene copy ratio was confirmed to be 1:1. CONCLUSIONS: The equivalence of pre-mRNA ratio level for wild-type and nonsense mutant in the rd/+ retina indicates that both genes were transcribed at similar levels. Thus, neither the nonsense mutation at position 347 nor the intron 1 retroviral insertion also present in the rd gene seem to have affected gene transcription. In contrast, the strain-independent bias favoring wild-type mature mRNA in vivo suggests a specific degradation of mutant transcript during or after pre-mRNA splicing. This allele-specific degradation serves to decrease mutant transcript levels dramatically in all rd strains, and suggests that photoreceptor cells have the capacity to reduce the level of an mRNA containing a nonsense mutation.

3',5'-Cyclic-GMP Phosphodiesterases↗

PubMed1997

Molecular cloning and characterization of CDEP, a novel human protein containing the ezrin-like domain of the band 4.1 superfamily and the Dbl homology domain of Rho guanine nucleotide exchange factors.

A cDNA for a novel human protein named CDEP was cloned using the subtractive hybridization method between dedifferentiated cartilage cells and overtly differentiated cartilage cells. CDEP cDNA contained an open reading frame encoding 1,045 amino acids in a total length of 3.4 kb. The deduced amino acid sequence revealed that a single polypeptide contained the ezrin-like domain, which is found in cytoskeleton-associated proteins of the band 4.1 superfamily, and the Dbl homology (DH) and pleckstrin homology (PH) domains, which are conserved in the Rho GEF (guanine nucleotide exchange factor) family. Northern blot analysis demonstrated that CDEP mRNA was expressed not only in the differentiated chondrocytes but also in various fetal and adult tissues. Since members of the band 4.1 superfamily and the Rho GEF family are crucial for microfilament organization, the novel protein CDEP may be involved in the adhesion, proliferation, and differentiation of some cell types including chondrocytes via changes in the cytoskeleton.

Amino Acid Sequence↗

PubMed1997

Proteolysis of the carboxyl-terminal GPI signal independent of GPI modification as a mechanism for selective protein secretion.

Variable amounts of soluble forms of a variety of glycosyl-phosphatidylinositol (GPI)-anchored proteins occur extracellularly, but the molecular mechanisms governing their release are not entirely clear. When the GPI-anchored folate receptor (FR) type beta was expressed transiently in human 293 fibroblasts, there was a roughly equal distribution of [3H]folic acid binding protein between the cell surface and the medium after 24 h over a wide range of expression levels of FR-beta. The difference in apparent molecular masses between the soluble FR-beta and the PI-PLC-treated membrane protein indicated that the former was not released from the membrane by the action of phospholipase. Brefeldin A inhibited the release of soluble FR-beta from both the transfected 293 cells and stable recombinant CHO (CHO-FR-beta) cells while pre-existing levels of cell surface FR were unaltered suggesting the absence of a precursor-product relationship between the membrane-associated FR-beta and the soluble protein in the medium. [35S]Cysteine pulse-chase analysis was consistent with this finding. Interchanging of carboxyl-terminal peptides between FR-beta and FR-alpha revealed that the nature of the processed signal for GPI modification was responsible for the quantitative membrane anchoring of FR-alpha and the production of soluble FR-beta. When total cell lysates were analyzed by Western blot, a diffuse band of apparent 41 kDa and three additional sharp bands of apparent 35, 33, and 29.3 kDa were seen. The 41 kDa band was identified as the PI-PLC sensitive cell surface receptor. Several mutant constructs of FR-beta, in which the carboxyl-terminal signal for GPI modification was either disrupted or deleted only gave the three lower bands. The three sharp bands from the wild-type and the mutant forms of FR-beta were identified as nonglycosylated (29.3 kDa) or glycosylated polypeptides in which the carboxyl-terminal peptide was at least partially proteolyzed without GPI modification. All of the mutations in the GPI signal resulted in the recovery of [3H]folic acid binding protein in the media which, similar to the wild-type FR recovered from the media, were converted to the 29.3 kDa band by N-glycanase. The results from this study indicate that a carboxyl-terminal peptide in FR-beta is efficiently proteolyzed intracellularly by a pathway that is independent of GPI signal recognition resulting in proper protein folding and secretion. Such carboxyl-terminal sequences could represent a simple adaptation for proteins whose physiologic functions reside both at the cell surface and in extracellular fluids, allowing their selective and tissue-specific release.

Amino Acid Sequence↗

PubMed1997

Neuronal dye coupling in the developing rat fascia dentata.

The present study describes dye coupling among neurons of the developing rat fascia dentata following impalement and intracellular filling with Neurobiotin. The number of neuronal impalements resulting in dye-coupled cells decreases from P14 to P120. The most rapid decline in dye coupling was observed between P14 and P21, the beginning of the most active period of synaptogenesis in the dentate molecular layer. Dye coupling between granule cells and axo-axonic interneurons (chandelier cells) accounts for about 10% of the dye-coupled neuronal population acquired in slices from P14 and P21 rats and declines to less than 5% by P60 and P120. Our data suggest that dye coupling is related reciprocally to the number of synapses formed on granule cells. Thus the relationship of dye coupling to synaptic density in the developing fascia dentata is similar to that reported in studies of the aging fascia dentata. Also the observation of axo-axonic interneurons coupled to granule cells at all ages suggests an interesting neuronal arrangement with the potential of limiting granule cell discharge to discrete neuronal assemblies in response to perforant path input.

Aging↗

PubMed1997

Stage-specific expression and phosphorylation of retinoblastoma protein (pRb) in the rat seminiferous epithelium.

To assess the potential role of retinoblastoma protein (pRb) in the regulation of cell cycle during spermatogenesis, the expression of retinoblastoma (Rb) mRNA and protein, as well as the phosphorylation states of pRb, in the rat seminiferous epithelial cycle, were studied. Two transcripts, 5.4 kb and 3.4 kb long, were detected in total RNA from the adult rat testis and only the 5.4 kb transcript was detected in poly (A)+-RNA from 8, 14 and 23-day old rat testes by Northern hybridization. Polysome analysis revealed that only a small portion of both Rb transcripts could be efficiently translated. By in situ hybridization, Rb mRNA was localized to germ cells from stage V pachytene spermatocytes to step 13 spermatids along the epithelial cycle. pRb immunoreactivity was detected in Sertoli cells and spermatogonia at all stages, as well as in the elongated steps 14-19 spermatids by immunohistochemistry. The amount of pRb and the phosphorylation status varied in a stage-specific manner in Western blots. These results show that pRb is expressed in the rat seminiferous epithelium in a cyclic fashion and suggest that it is involved in the regulation of proliferation of spermatogonia and maintenance of the differentiation status of Sertoli cells and spermatids.

Animals↗

PubMed1997

Binding of high molecular weight kininogen to human endothelial cells is mediated via a site within domains 2 and 3 of the urokinase receptor.

The urokinase receptor (uPAR) binds urokinase-type plasminogen activator (u-PA) through specific interactions with uPAR domain 1, and vitronectin through interactions with a site within uPAR domains 2 and 3. These interactions promote the expression of cell surface plasminogen activator activity and cellular adhesion to vitronectin, respectively. High molecular weight kininogen (HK) also stimulates the expression of cell surface plasminogen activator activity through its ability to serve as an acquired receptor for prekallikrein, which, after its activation, may directly activate prourokinase. Here, we report that binding of the cleaved form of HK (HKa) to human umbilical vein endothelial cells (HUVEC) is mediated through zinc-dependent interactions with uPAR. These occur through a site within uPAR domains 2 and 3, since the binding of 125I-HKa to HUVEC is inhibited by vitronectin, anti-uPAR domain 2 and 3 antibodies and soluble, recombinant uPAR (suPAR), but not by antibody 7E3, which recognizes the beta chain of the endothelial cell vitronectin receptor (integrin alphavbeta3), or fibrinogen, another alphavbeta3 ligand. We also demonstrate the formation of a zinc-dependent complex between suPAR and HKa. Interactions of HKa with endothelial cell uPAR may underlie its ability to promote kallikrein-dependent cell surface plasmin generation, and also explain, in part, its antiadhesive properties.

Cells, Cultured↗

PubMed1997

Molecular characterization of the novel basic helix-loop-helix protein DEC1 expressed in differentiated human embryo chondrocytes.

The differentiation of human embryo chondrocytes was markedly induced by the addition of Bt2cAMP to the culture medium. Using this culture system, a novel human cDNA for a basic helix-loop-helix (bHLH) protein (named DEC1) expressed primarily in the chondrocytes in response to Bt2cAMP was cloned by the subtractive hybridization method. DEC1 protein consists of 412 amino acids and exhibits structural similarities to the mammalian HES family, Drosophila hairy, and Enhancer of split m7 in the bHLH region. Northern blot analysis showed that DEC1 mRNA was expressed in various tissues including the cartilage, lung, spleen, and intestine, but not in the brain. These findings suggest that the bHLH factor DEC is involved in the control of cell differentiation in several tissues including cartilage.

Amino Acid Sequence↗

PubMed1997

High molecular weight kininogen peptides inhibit the formation of kallikrein on endothelial cell surfaces and subsequent urokinase-dependent plasmin formation.

A sequence of 31 amino acids (S565-K595) in domain 6 of the light chain of high molecular weight kininogen (HK) has previously been shown to be responsible for the binding of plasma prekallikrein (PK) or kallikrein. To find effective peptides that might block binding between HK and PK on cell surfaces, a new series of synthetic peptides has now been prepared that incorporates portions of this binding domain sequence. For mapping the minimal sequence within HK, these new peptides were tested for their ability to compete with HK for binding PK in a cell-free system and on human umbilical vein endothelial cells (HUVEC). In the former, at pH 7.4, the kds for binding between kallikrein and either D567-K595, S565-P594, D567-S593, or D567-T591 were all similar to that for the binding of S565-K595 (0.2 to 0.4 micromol/L), but those for the binding of D568-K595, W569-K595, and D567-P589 were an order of magnitude greater (kd = 2 to 5 micromol/L). D567-S586, the shortest chain length of the N- and C-terminal truncation sequences tested, does not effectively compete with kininogen for kallikrein binding (kd = 100 micromol/L). These results imply that D567-T591, a 25-residue peptide (HK25c), contains sufficient structural information for binding kallikrein in solution. D567-T591 also is the minimum structural sequence to block binding of kallikrein to HUVEC-bound HK (IC50 = 50 nmol/L) and to inhibit PK activation to kallikrein on the cell surface (IC50 = 80 nmol/ L). In addition, D567-T591 also inhibits the generation of kallikrein-activated urokinase, which activates plasminogen to plasmin (IC50 = 100 nmol/L). Thus, HK-derived peptides may be useful compounds for modulating excessive fibrinolysis and hypotension in sepsis and multiple trauma.

Amino Acid Sequence↗

PubMed1997

Effects of concanavalin A on chondrocyte hypertrophy and matrix calcification.

Resting chondrocytes do not usually undergo differentiation to the hypertrophic stage and calcification. However, incubating these cells with concanavalin A resulted in 10-100-fold increases in alkaline phosphatase activity, binding of 1,25(OH)2-vitamin D3, type X collagen synthesis, 45Ca incorporation into insoluble material, and calcium content. On the other hand, other lectins tested (including wheat germ agglutinin, lentil lectin, pea lectin, phytohemagglutinin-L, and phytohemagglutinin-E) marginally affected alkaline phosphatase activity, although they activate lymphocytes. Methylmannoside reversed the effect of concanavalin A on alkaline phosphatase within 48 h. Concanavalin A did not increase alkaline phosphatase activity in articular chondrocyte cultures. In resting chondrocyte cultures, succinyl concanavalin A was as potent as concanavalin A in increasing alkaline phosphatase activity, the incorporation of [35S]sulfate, D-[3H]glucosamine, and [3H]serine into proteoglycans, and the incorporation of [3H]serine into protein, although concanavalin A, but not succinyl concanavalin A, induced a rapid change in the shape of the cells from flat to spherical. These findings suggest that concanavalin A induces a switch from the resting, to the growth-plate stage, and that this action of concanavalin A is not secondary to changes in the cytoskeleton. Chondrocytes exposed to concanavalin A may be useful as a novel model of endochondral bone formation.

Alkaline Phosphatase↗

PubMed1997

Characterization of a cartilage-derived 66-kDa protein (RGD-CAP/beta ig-h3) that binds to collagen.

A 66-kDa collagen fiber-associated protein (RGD-CAP) was isolated from a fiber-rich fraction of pig cartilage by ultrafiltration and collagen-affinity chromatography. Amino acid sequencing and cDNA cloning indicated that the RGD-CAP is identical or closely related to beta ig-h3 protein which is induced in human adenocarcinoma cells by transforming growth factor-beta (TGF-beta) (Skonier, J., Neubauer, M., Madisen, L., Bennett, K., Plowman, G.D., and Purchio, A.F. (1992) DNA Cell. Biol. 11, 511-522). The RGD-CAP, as well as beta ig-h3, has the RGD sequence in the C-terminal region. The native RGD-CAP bound to type I, II, and IV collagens even in the presence of 1 M NaCl. A recombinant preparation of RGD-CAP expressed in Escherichia coli cells also bound to collagen but not to gelatin. The RGD-CAP mRNA was expressed in chondrocytes throughout all stages, although the expression level was highest during the prehypertrophic stage. In addition, TGF-beta increased the RGD-CAP mRNA level in chondrocyte cultures. Since RGD-CAP transcripts were found in most tissues, this novel collagen-binding protein may play an important role in cell-collagen interactions in various tissues including developing cartilage.

Amino Acid Sequence↗