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Biomedical subjects

H C Wu

Publications and source records attributed to H C Wu.

At least 55 records · Page 3Linked to original sources

G protein beta subunit is closely associated with microtubules.

Previously, we have identified the association of G protein beta subunit (Gbeta) with mitotic spindles in various mammalian cells. Since microtubules are the main component of mitotic spindles, here we have isolated bovine brain microtubules and purified Gbeta subunit to identify the close association of Gbeta subunit with purified brain microtubules and have shown the direct incorporation of Gbeta subunit into the microtubules both in vitro and in vivo. It was found that: (1) microtubular fraction isolated from bovine brain contained Gbeta subunit, (2) coimmunoprecipitation demonstrated that Gbeta subunit could be coprecipitated with tubulin, (3) addition of purified Gbeta subunit into cytosolic extract for microtubule assembly caused direct incorporation of Gbeta subunit into assembled microtubules and increased the association of microtubule-associated proteins with microtubules, and (4) incubation of exogenous Gbeta subunit with detergent-permeabilized cells resulted in direct incorporation of Gbeta subunit into microtubule fibers and depolymerized tubulin molecules. We conclude that G protein beta subunit is closely associated with microtubules and may play an important role in the regulation of microtubule formation in addition to its regulatory role in cellular signal transduction.

Animals↗

Transcription activation by Escherichia coli FNR protein: similarities to, and differences from, the CRP paradigm.

During transcription activation at FNR-dependent promoters where the DNA site for FNR overlaps the -35 element, a surface-exposed activating region in the upstream subunit of the FNR dimer interacts with the C-terminal domain of the RNA polymerase alpha subunit. Starting with a cloned fnr gene encoding a defective FNR derivative carrying substitutions in this activating region, we screened a library of random mutations to identify substitutions that restored FNR activity. Activity can be restored by substitutions at residues T118, E47 and K60. The locations of these residues identify three separate surface-exposed regions of FNR that can play a role in transcription activation. These three regions appear to be analogues of Activating Region 1, Activating Region 2 and Activating Region 3 of the cyclic AMP receptor protein, CRP: our results underscore the similarities between FNR and CRP.

Bacterial Proteins↗

Inhibition of the cytotoxicity of protein toxins by a novel plant metabolite, mansonone-D.

We have studied the effect of several structurally related mansonones on the cytotoxicity of plant and bacterial toxins in Vero and BER-40, a brefeldin A-resistant mutant of Vero cells. Mansonone-D (MD), a sesquiterpenoid ortho-naphthoquinone, inhibited the cytotoxicity of ricin, modeccin, Pseudomonas toxin, and diphtheria toxin in Vero cells to different extents. The inhibition of ricin cytotoxicity was dose dependent and reversed upon removal of the drug. Protection of ricin cytotoxicity was also observed in the presence of cycloheximide, indicating that de novo protein synthesis is not required for the protective effect. Although MD inhibited the degradation and excretion of ricin, the binding and internalization of ricin was not affected. In contrast, MD strongly reduced the specific binding of diphtheria toxin in Vero cells. Fluorescence microscopic studies show that MD treatment dramatically alters the morphology of the Golgi apparatus in Vero cells. The kinetic studies reveal that the protection of ricin cytotoxicity is the consequence of decreased toxin translocation to the cytosol in MD-treated cells. The reactive ortho-quinone moiety of MD is important for the protective effect as thespesone, a para-naphthoquinone with a heterocyclic ring structure identical to that of MD, did not inhibit the cytotoxicity of toxins. Thespone, a dehydromansonone-D, lacking two hydrogens from the heterocyclic dihydrofuran ring of MD, inhibited the cytotoxicity of ricin, but was albeit less potent than MD. Neither mansonone-E nor mansonone-H with reactive ortho-quinone moiety, but with a different heterocyclic structure, had any effect on the cytotoxicity of ricin indicating that the protective effect of MD is specifically related to the overall structure of the metabolite.

Animals↗

Brain-derived neurotrophic factor antisense oligonucleotide impairs memory retention and inhibits long-term potentiation in rats.

We have examined the relationship between brain-derived neurotrophic factor gene expression in the hippocampus and memory retention as well as long-term potentiation of rats. One-way inhibitory avoidance learning was adopted as the behavioural paradigm. Results revealed that brain-derived neurotrophic factor messenger RNA levels in the dentate gyrus of the hippocampus were markedly increased at 1 h, 3 h and 6 h post-training in rats showing good retention performance when compared with the poor retention controls. Direct injection of brain-derived neurotrophic factor antisense oligonucleotide into the dentate gyrus of the hippocampus before memory consolidation takes place markedly impaired retention performance in rats. It also significantly decreased brain-derived neurotrophic factor messenger RNA level in the dentate gyrus. The same antisense treatment also markedly reduced the amplitude and slope of excitatory postsynaptic potential as well as the brain-derived neurotrophic factor messenger RNA level in the dentate gyrus. These results suggest that hippocampal brain-derived neurotrophic factor gene expression plays an important role in the memory consolidation process and in the expression of long-term potentiation in rats. These results provide the first evidence to relate brain-derived neurotrophic factor gene expression and memory function in vertebrates. It further suggests that brain-derived neurotrophic factor gene expression is involved in behavioural plasticity.

Animals↗

Identification of a rat brain gene associated with aging by PCR differential display method.

The polymerase chain reaction (PCR) differential display method is a powerful tool to detect and characterize alteration of gene expression in eukaryotic cells. In order to screen the differentially expressed genes between the adult (3 mo) and aged (24 mo) rats, the PCR differential display method was adopted in the present study. One differentially expressed cDNA band (C7-1) was identified and the aged rats expressed more the C7-1 gene than the adult rats in the frontal cortex, but not in the hippocampus and hypothalamus. The C7-1 cDNA band was recovered, reamplified, and subcloned as a probe in Northern blot analysis. A transcript of approx 2.8 kb was expressed in the frontal cortex of both the adult and aged rats, but the C7-1 mRNA level was increased for 52% in the aged rats. The C7-1 gene was then sequenced that contains 243 bp. We have found that the C7-1 cDNA shows no significant homology to any published genes, suggesting that the C7-1 gene is an unknown gene associated with aging. This study provides the first evidence to show that there is alteration in gene expression associated with aging by using the PCR differential display method.

Aging↗

Antisense oligonucleotides to corticotropin-releasing factor impair memory retention and increase exploration in rats.

Corticotropin-releasing factor is known as a stress peptide which stimulates adrenocorticotropin and corticosterone release. It also has a direct central effect to produce various behavioural activations in rats. In the present study, we have designed and studied the effects of an antisense oligonucleotide directed against the corticotropin-releasing factor gene in rats. Local injection of this antisense oligonucleotide into the hippocampus (1 nmol, five injections) significantly impaired the retention performance of an inhibitory avoidance task in rats. In another experiment, the same injection (1 nmol, four injections) also markedly increased rearing response and total distance travelled by rats in a novel activity chamber. Meanwhile, it markedly decreased grooming behaviour in rats. When directly injected into the hypothalamus in another group of animals (1 nmol, four injections), this antisense oligonucleotide significantly decreased the plasma adrenocorticotropin level. The effectiveness of these antisense oligonucleotide treatments was verified by a significant reduction in the level of corticotropin-releasing factor messenger RNA in specific brain regions as determined by quantitative reverse transcription polymerase chain reaction analysis. These results strengthen the importance of corticotropin-releasing factor in modulating memory processing and exploratory behaviour in rats. This work also provides an important tool for studying other physiological functions that corticotropin-releasing factor may mediate or modulate in mammals.

Adrenocorticotropic Hormone↗

A Salmonella typhimurium genetic locus which confers copper tolerance on copper-sensitive mutants of Escherichia coli.

Three distinct clones from a Salmonella typhimurium genomic library were identified which suppressed the copper-sensitive (Cu(s)) phenotype of cutF mutants of Escherichia coli. One of these clones, pCUTFS2, also increased the copper tolerance of cutA, -C, and -E mutants, as well as that of a lipoprotein diacylglyceryl transferase (lgt) mutant of E. coli. Characterization of pCUTFS2 revealed that the genes responsible for suppression of copper sensitivity (scs) reside on a 4.36-kb DNA fragment located near 25.4 min on the S. typhimurium genome. Sequence analysis of this fragment revealed four open reading frames (ORF120, ORF627, ORF207, and ORF168) that were organized into two operons. One operon consisted of a single gene, scsA (ORF120), whereas the other operon contained the genes scsB (ORF627), scsC (ORF207), and scsD (ORF168). Comparison of the deduced amino acid sequences of the predicted gene products showed that ScsB, ScsC, and ScsD have significant homology to thiol-disulfide interchange proteins (CutA2, DipZ, CycZ, and DsbD) from E. coli and Haemophilus influenzae, to an outer membrane protein (Com1) from Coxiella burnetii, and to thioredoxin and thioredoxin-like proteins, respectively. The two operons were subcloned on compatible plasmids, and complementation analyses indicated that all four proteins are required for the increased copper tolerance of E. coli mutants. In addition, the scs locus also restored lipoprotein modification in lgt mutants of E. coli. Sequence analyses of the S. typhimurium scs genes and adjacent DNAs revealed that the scs locus is flanked by genes with high homology to the cbpA (predicted curved DNA-binding protein) and agp (acid glucose phosphatase) genes of E. coli located at 22.90 min (1,062.07 kb) and 22.95 min (1,064.8 kb) of the E. coli chromosome, respectively. However, examination of the E. coli chromosome revealed that these genes are absent at this locus and no evidence has thus been obtained for the occurrence of the scs locus elsewhere on the genome.

Amino Acid Sequence↗

Roles of histidine-103 and tyrosine-235 in the function of the prolipoprotein diacylglyceryl transferase of Escherichia coli.

Phosphatidylglycerol:prolipoprotein diacylglyceryl transferase (Lgt) is the first enzyme in the posttranslational sequence of reactions resulting in the lipid modification of lipoproteins in bacteria. A previous comparison of the primary sequences of the Lgt enzymes from phylogenetically distant bacterial species revealed several highly conserved amino acid sequences throughout the molecule; the most extensive of these was the region 103HGGLIG108 in the Escherichia coli Lgt (H.-Y. Qi, K. Sankaran, K. Gan, and H. C. Wu, J. Bacteriol. 177:6820-6824, 1995). These studies also revealed that the kinetics of inactivation of E. coli Lgt with diethylpyrocarbonate were consistent with the modification of a single essential histidine or tyrosine residue. The current study was conducted in an attempt to identify this essential amino acid residue in order to further define structure-function relationships in Lgt. Accordingly, all of the histidine residues and seven of the tyrosine residues of E. coli Lgt were altered by site-directed mutagenesis, and the in vitro activities of the altered enzymes, as well the abilities of the respective mutant lgt alleles to complement the temperature-sensitive phenotype of E. coli SK634 defective in Lgt activity, were determined. The data obtained from these studies, in conjunction with additional chemical inactivation studies, support the conclusion that His-103 is essential for Lgt activity. These studies also indicated that Tyr-235 plays an important role in the function of this enzyme. Although other histidine and tyrosine residues were not found to be essential for Lgt activity, alterations of His-196 resulted in a significant reduction of in vitro activity.

Amino Acid Sequence↗

The clinical experience of gaseous retroperitoneoscopic and gasless retroperitoneoscopy-assisted unroofing of renal cyst.

BACKGROUND: The aim of this study is to compare the application of gaseous retroperitoneoscopic (GR) and gasless retroperitoneoscopy-assisted (GLRA) unroofing of renal cysts. METHODS: Fourteen patients with symptomatic simple renal cysts had undergone unroofing of the cyst with GR in seven cases and GLRA in seven others. Three trocars (10 mm, 10 mm and 5 mm) were inserted in the GR procedure. A 3 cm flank muscle-split incision was made and retroperitoneoscopy was performed through the same incision in the GLRA procedure. Then, the cyst was unroofed. RESULTS: The mean operative time was 104.3 minutes in the GR group and 52.1 minutes in the GLRA group, respectively (p = 0.001). The mean requirement of postoperative meperidine hydrochloride injection was 21.4 mg in the GR group and 71.4 mg in the GLRA group, respectively (p = 0.017). In the GR group, the mean postoperative stay was 3.7 days, and the time needed for return to normal activity was 7 days. In the GLRA group, the mean postoperative stay was 4.6 days, and the time needed for return to normal activity was 8 days. CONCLUSIONS: GR and GLRA techniques for unroofing of renal cysts are safe, effective and minimally invasive. GLRA is easy to perform and a more time-saving procedure when compared to GR, however, the patients of GLRA suffered more postoperative pain than after GR. GLRA is recommended in patients who had received retroperitoneal surgery or who have multiple renal cysts.

Adult↗

Prognostic factors of primary aldosteronism.

BACKGROUND: Primary aldosteronism (PA) is a rare but potentially curable cause of hypertension. Between October 1982 and November 1994, 30 patients of PA received unilateral adrenalectomy with a long-term follow up (mean:60.3 months). Nineteen (63.3%) cases were cured (Group 1); 11(36.7%) cases were improved (Group 2). The purpose of this study was to determine prognostic factors after surgery in patients with PA. METHODS: A retrospective analysis was performed regarding age and sex of the patients, duration of hypertension, family history of hypertension, preoperative blood pressure, plasma aldosterone concentration (PAC), plasma renin activity (PRA) and efficacy of spironolactone on blood pressure between both groups. End-organs (including kidney, heart, retina and brain) involvement was evaluated and compared. Adrenalectomy and renal biopsy specimen for pathology were similarly evaluated. RESULTS: The duration of hypertension was longer in Group 2 than in Group 1 (8.18 +/- 4.94 vs 5.21 +/- 4.24 years). The efficacy of spironolactone on blood pressure (BP) was positive in 81.8% of Group 1 and 16.7% of Group 2. Adrenal cortical adenoma in 24 cases with a cure rate of 70.8% (17/24) and adrenal cortical macronodular hyperplasia in 6 cases with a cure rate of 33.3% (2/6) were noted. Group 2 had more end-organs involvement than Group 1. The severity of histopathological change of the renal biopsy was similar. CONCLUSIONS: This study suggests that preoperative response of blood pressure to spironolactone administration predicts the postoperative prognosis of hypertension in patient with PA. Long duration of hypertension and involvement of two or more end-organs were poor prognostic factors. Excellent results can be achieved by unilateral adrenalectomy in adrenal cortical adenoma and fair results, in adrenal cortical macronodular hyperplasia.

Adolescent↗

Ceramide reverses brefeldin A (BFA) resistance in BFA-resistant cell lines.

We have found that C6 ceramide, a cell-permeable ceramide analog, partially restored the brefeldin A (BFA) sensitivity in a BFA-resistant mutant of Vero cells (BER-40) and in the naturally BFA-resistant Madin-Darby canine kidney (MDCK) cells. Incubation of BER-40 and MDCK cells with low concentrations of C6 ceramide resulted in (i) a pronounced increase in BFA cytotoxicity as measured by the inhibition of [3H]thymidine incorporation and the inhibition of colony formation by BFA, (ii) a significant protection by BFA against ricin cytotoxicity, and (iii) an inhibition of bulk protein secretion by BFA in BER-40 and MDCK cells. Related sphingolipids including sphingosine, sphingomyelin, and lactosylceramide and other unrelated lipid second messengers such as arachidonic acid and 1,2-diacylglycerol did not elicit the protection of BER-40 and MDCK cells against ricin cytotoxicity by BFA. C6 ceramide was the most effective among the ceramides with different acyl chain lengths. Interestingly, dihydro-C6 ceramide, which lacks the trans double bond in the sphingoid base, had no effect. On the other hand, C6 ceramide did not enhance BFA sensitivity in BFA-sensitive Vero cells. The LD50 of C6 ceramide were similar in Vero and BER-40 cells. Fluorescence microscopic studies revealed that C6 ceramide induced the redistribution of beta-COP from the Golgi membranes to a more dispersed localization in both BFA-sensitive and BFA-resistant cell lines, mimicking the effect of BFA. Suboptimal concentration of C6 ceramide also restored the effect of BFA on the beta-COP distribution in BER-40 and MDCK cells. These results indicate that C6 ceramide restores the BFA sensitivity in BFA-resistant BER-40 and MDCK cells.

Animals↗

Ilimaquinone inhibits the cytotoxicities of ricin, diphtheria toxin, and other protein toxins in Vero cells.

Ilimaquinone (IQ), a metabolite from sea sponges, has been shown to cause the breakdown of Golgi membranes into small vesicular structure and to inhibit protein transport without eliciting the retrograde transport of the Golgi enzymes to the endoplasmic reticulum [P. A. Takizawa, J. K. Yucel, B. Viet, D. J. Faulkner, T. Deerinck, G. Soto, M. Ellismann, and V. Malhotra, Cell (1993) 73, 1079-1090]. We have found that incubation of Vero cells with IQ inhibited the cytotoxicity of ricin in a dose-dependent manner. The inhibition was reversed upon the removal of IQ. Neither binding and internalization of 125I-ricin nor the translocation of ricin to the cytosol was affected by IQ. However, IQ significantly inhibited the recycling and degradation of internalized 125I-ricin. Preincubation with IQ also prevented the enhancement of ricin cytotoxicity by NH4Cl or nigericin. The inhibition of ricin cytotoxicity by IQ was observed in the presence of cycloheximide, indicating that de novo protein synthesis is not required for IQ-mediated protection of Vero cells from ricin cytotoxicity. In contrast to perinuclear distribution of TRITC-labeled ricin in Vero cells, TRITC-ricin appeared in numerous small vesicles dispersed throughout the cytoplasm in IQ-treated Vero cells. Double labeling with C6-NBD-ceramide and TRITC-labeled ricin showed that these ricin-containing vesicles were distinct from the IQ-induced breakdown product of the Golgi membranes. Like brefeldin A (BFA), IQ inhibited the cytotoxicities of abrin, modeccin, Pseudomonas toxin, and Shiga-like toxin in Vero cells. Unlike BFA, IQ also inhibited the cytotoxicity of diphtheria toxin (DT). Inhibition of DT cytotoxicity was the consequence of a decreased specific binding of the toxin in the IQ-treated cells.

Animals↗

Protective effect of cell-permeable ceramide analogs against modeccin, ricin, Pseudomonas toxin, and diphtheria toxin.

We investigated the effects of various ceramide (Cer) analogs and related sphingolipids on the cytotoxicities of modeccin, ricin, Pseudomonas toxin, and diphtheria toxin in various cell lines. The most pronounced protective effect by C6Cer, a short-chain cell-permeable Cer analog, was observed in modeccin cytotoxicity in Vero, BER-40, and MDCK cells, whereas the cytotoxicity of diphtheria toxin was not affected by any of the ceramide analogs tested. C6Cer did not affect the binding and internalization of ricin and modeccin in Vero and BER-40 cells. C2Cer and C8Cer also protected against modeccin cytotoxicity, albeit less effectively than C6Cer. However, related sphingolipids including sphingosine, sphingomyelin, lactosylceramide, C18Cer (the naturally occurring ceramide), and dihydro C6Cer had no effect. A correlation was found between the ability of ceramides to inhibit bulk protein secretion and the inhibition of modeccin cytotoxicity by ceramides. Among Cer analogs tested, C6Cer, the most potent inhibitor of modeccin cytotoxicity, strongly inhibited bulk protein secretion in Vero, BER-40, and MDCK cells. PtK1 cells, which were not protected by ceramides against toxins, were resistant to ceramide-induced inhibition of bulk protein secretion. These results confirm that Cer may modulate the intracellular transport of proteins through the Golgi complex. Such Cer-sensitive processes may be involved in the intoxication of cells by plant and bacterial toxins, especially modeccin.

Animals↗

Ability of the Atherosclerosis Risk in Communities (ARIC)/Baecke Questionnaire to assess leisure-time physical activity.

BACKGROUND: The Atherosclerosis Risk in Communities (ARIC)/Baecke Questionnaire is a general survey of both occupational and leisure (sport and exercise related and non-sport and exercise related) physical activity. METHODS: Its ability to assess leisure physical activity was studied in 78 men and women, age 20-59 years, by comparison to: six 48-hour physical activity records; 14 48-hour Caltrac accelerometer readings (Caltrac); three peak oxygen consumption (VO2 peak) determinations; and per cent body fat. These criteria measures were obtained over a year's duration. RESULTS: The following associations were evident in men and women respectively, between sport and exercise physical activity and: physical activity record heavy intensity activity (r = 0.73 and r = 0.63); VO2 peak (r = 0.67 and r = 0.45); and per cent body fat (r = -0.37, P = 0.08 and r = -0.44). Less concordance was evident (r = 0.39 for men and r = 0.23, NS, for women) between non-sport and exercise physical activity and physical activity record light intensity activity. Questionnaire and physical activity record indices of total leisure activity tended to be more closely related in men (r = 0.59) than women (r = 0.33). For both men and women, survey results were not closely associated with Caltrac readings. CONCLUSIONS: Although there are weaknesses, questionnaire strengths consistent for both men and women include: ease of administration, high reliability, and accurate assessment of heavy intensity activity as well as light intensity activities such as walking and bicycling.

Adipose Tissue↗

Identification of cutC and cutF (nlpE) genes involved in copper tolerance in Escherichia coli.

It has been suggested previously that copper transport in Escherichia coli is mediated by the products of at least six genes, cutA, cutB, cutC, cutD, cutE, and cutF. A mutation in one or more of these genes results in an increased copper sensitivity (D. Rouch, J. Camakaris, and B. T. O. Lee, p. 469-477, in D. H. Hamer and D. R. Winge, ed., Metal Ion Homeostasis: Molecular Biology and Chemistry, 1989). Copper-sensitive cutC and cutF mutants were transformed with a genomic library of E. coli, and copper-tolerant transformants were selected. Two distinct clones were identified, each of which partially restores copper tolerance in both the cutC and cutF mutants of E. coli. Subcloning, physical mapping, and sequence analysis have revealed that the cutC gene is located at 42.15 min on the E. coli genome and encodes a cytoplasmic protein of 146 amino acids and that the cutF gene is located at 4.77 min on the E. coli genome and is allelic to the nlpE gene independently identified by Silhavy and coworkers (W. B. Snyder, L. J. B. Davis, P. N. Danese, C. L. Cosma, and T. J. Silhavy, J. Bacteriol. 177:4216-4223, 1995). Results from the genetic mapping of the copper-sensitive mutations in the cutF mutant and sequencing of the cutC and cutF (nlpE) alleles from both cutC and cutF mutants indicate that both the cutC and cutF mutants are in fact double mutants altered in these two genes, and mutations in both the genes appear to be required for the copper-sensitive phenotype in each mutant.

Alleles↗

Structure-function relationship of bacterial prolipoprotein diacylglyceryl transferase: functionally significant conserved regions.

The structure-function relationship of bacterial prolipoprotein diacylgyceryl transferase (LGT) Has been investigated by a comparison of the primary structures of this enzyme in phylogenetically distant bacterial species, analysis of the sequences of mutant enzymes, and specific chemical modification of the Escherichia coli enzyme. A clone containing the gene for LGT, lgt, of the gram-positive species Staphylococcus aureus was isolated by complementation of the temperature-sensitive lgt mutant of E. coli (strain SK634) defective in LGT activity. In vivo and in vitro assays for prolipoprotein diacylglyceryl modification activity indicated that the complementing clone restored the prolipoprotein modification activity in the mutant strain. Sequence determination of the insert DNA revealed an open reading frame of 837 bp encoding a protein of 279 amino acids with a calculated molecular mass of 31.6 kDa. S. aureus LGT showed 24% identity and 47% similarity with E. coli, Salmonella typhimurium, and Haemophilus influenzae LGT.S. aureus LGT, while 12 amino acids shorter than the E. coli enzyme, had a hydropathic profile and a predicted pI (10.4) similar to those of the E. coli enzyme. Multiple sequence alignment among E. coli, S. typhimurium, H. influenzae, and S. aureus LGT proteins revealed regions of highly conserved amino acid sequences throughout the molecule. Three independent lgt mutant alleles from E. coli SK634, SK635, and SK636 and one lgt allele from S. typhimurium SE5221, all defective in LGT activity at the nonpermissive temperature, were cloned by PCR and sequenced. The mutant alleles were found to contain a single base alteration resulting in the substitution of a conserved amino acid. The longest set of identical amino acids without any gap was H-103-GGLIG-108 in LGT from these four microorganisms. In E. coli lgt mutant SK634, Gly-104 in this region was mutated to Ser, and the mutant organism was temperature sensitive in growth and exhibited low LGT activity in vitro. Diethylpyrocarbonate inactivated the E. coli LGT with a second-order rate constant of 18.6 M-1S-1, and the inactivation of LGT activity was reversed by hydroxylamine at pH 7. The inactivation kinetics were consistent with the modification of a single residue, His or Tyr, essential for LGT activity.

Amino Acid Sequence↗