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P F Bray

Publications and source records attributed to P F Bray.

At least 55 records · Page 3Linked to original sources
PubMed1995

Overexpression of cyclin D1 in the Dami megakaryocytic cell line causes growth arrest.

The maturation of megakaryocytes in vivo requires polyploidization or repeated duplication of DNA without cytokinesis. As DNA replication and cytokinesis are tightly regulated in somatic cells by cyclins and cyclin-dependent kinases, we sought to determine the pattern of cyclin gene expression in cells that undergo megakaryocytic differentiation and polyploidization. The Dami megakaryocytic cell line differentiates and increases ploidy in response to phorbol 12-myristate 13-acetate (PMA) stimulation in vitro. We used Northern blotting to analyze mRNA levels of cyclins A, B, C, D1, and E in PMA-induced Dami cells and found that cyclin D1 mRNA levels increased dramatically (18-fold). Similar increases in cyclin D1 mRNA were obtained for other cell lines (HEL and K562) with megakaryocytic properties, but not in HeLa cells. The increase in cyclin D1 was confirmed by Western immunoblotting of PMA-treated Dami cells. This finding suggested that cyclin D1 might participate in megakaryocyte differentiation by promoting endomitosis and/or inhibiting cell division. To address these possibilities, we constructed two stable Zn+2-inducible, cyclin D1-overexpressing Dami cell lines. Cyclin D1 expression alone was not sufficient to induce polyploidy, but in conjunction with PMA-induced differentiation, polyploidization was slightly enhanced. However, unlike other cell systems, cyclin D1 overexpression caused cessation of cell growth. Although the mechanism by which cyclin D1 may affect megakaryocyte differentiation is not clear, these data demonstrate that cyclin D1 is upregulated in differentiating megakaryocytic cells and may contribute to differentiation by arresting cell proliferation.

Cell Cycle↗
PubMed1995

Molecular genotyping of fetal platelet antigens with uncultured amniocytes.

OBJECTIVE: Amino acid substitutions in platelet membrane glycoproteins result in alloantigens implicated in neonatal alloimmune thrombocytopenia. We report the use of the reverse dot blot technique to genotype the five major fetal platelet alloantigens from amniotic fluid cells. STUDY DESIGN: We evaluated a patient with Bakb platelet antibodies who had a previous pregnancy complicated by fetal intracranial hemorrhage. The father was heterozygous Baka/Bakb, giving the pregnancy a 50% risk for platelet incompatibility between mother and fetus. Amniotic fluid was obtained at 16 weeks. Deoxyribonuleic acid was extracted from uncultured amniocytes and amplified with polymerase chain reaction. These products were hybridized to filters containing oligonucleotides specific for each of the 10 different platelet antigen alleles. Reactivity was detected with a chromogenic substrate. RESULTS: The reverse dot blot genotyping of uncultured amniocytes revealed the fetus to be Baka/Baka, thus not at risk for neonatal alloimmune thrombocytopenia. CONCLUSION: Precise knowledge of fetal platelet type by amniocentesis could obviate the need for fetal blood sampling and significantly alter prenatal management of neonatal alloimmune thrombocytopenia.

Adult↗
PubMed1995

Gene frequencies of the five major human platelet antigens in African American, white, and Korean populations.

BACKGROUND: The study of the immunogenetics of the human platelet antigens is important to the improvement of diagnosis and genetic counseling and to the development of screening programs for women at risk of having babies with neonatal alloimmune thrombocytopenia. Description of the immunogenetics of the human platelet antigens in some racial groups has been incomplete. STUDY DESIGN AND METHODS: A reverse dot blot technique employing polymerase chain reaction-amplified genomic DNA was applied in genotyping the five major human platelet antigens in the following populations: 100 African American and 100 white women admitted to the obstetric unit at Johns Hopkins Hospital (Baltimore, MD) and 100 inpatients at Yonsei University (Seoul, Korea). RESULTS: The gene frequencies of HPA-2b (Koa) and HPA-5b (Bra) in African Americans were twice those in whites (African Americans: 0.18 and 0.21, respectively; whites: 0.09 and 0.11, respectively). There is a very low gene frequency of the HPA-1b (PIA2) allele in Koreans (0.005). No significant differences were found in the gene frequencies of the human platelet antigens in whites in this series and those in published European studies. CONCLUSION: These studies indicate a higher potential risk for alloimmunization to HPA-2 (Ko) and HPA-5 (Br) antigens in African Americans than in whites. In addition, the low gene frequency of HPA-1b (PIA2) in African Americans and Koreans suggests that alloimmunization to HPA-1a (PIA1) would be very unusual in these populations. These data may provide the basis for planning neonatal alloimmune thrombocytopenia screening programs in certain ethnic populations.

Antigens, Human Platelet↗
PubMed1995

A monoclonal antibody (SZ21) specific for platelet GPIIIa distinguishes P1A1 from P1A2.

Neonatal alloimmune thrombocytopenia (NATP) and post-transfusion purpura (PTP) are acquired bleeding disorders caused by alloimmune thrombocytopenia. In most cases, the thrombocytopenia is due to an alloantibody directed against the platelet glycoprotein IIb-IIIa (GPIIb-IIIa) complex. During the course of routine studies on the role of GPIIb-IIIa in inherited and acquired bleeding and thrombotic disorders, we unexpectedly identified an individual whose platelets reacted by non-reduced Western blot analysis with anti-GPIIIa polyclonal antisera, but did not react with a commercially available monoclonal antibody (SZ21) specific for GPIIIa. We screened all 14 GPIIIa exons for possible nucleotide changes which might alter amino acids and found variations in only exons 3 and 10. Nucleotide sequencing revealed that only the exon 3 alteration changed the predicted amino acid sequence. This variation was caused by homozygosity for the uncommon P1A2 allele of the GPIIIa gene. Platelets from two additional unrelated normal individuals known to be homozygous for P1A2 also lacked reactivity with SZ21 by Western blot. Using flow cytometry with intact platelets, we observed a markedly reduced binding of SZ21 to platelets with the P1A2 genotype. Scatchard analyses indicated that SZ21 bound to P1A1/A1 platelets with a Kd of approximately 8.26 x 10(-10) M, and to P1A2/A2 platelets with a Kd of approximately 5.58 x 10(-9) M. Thus, we have characterized a readily available monoclonal antibody able to distinguish between the two P1A alleles of the GPIIIa gene. Because incompatibility for this platelet polymorphism is the most common cause of neonatal alloimmune thrombocytopenia and posttransfusion purpura, and because platelet immunophenotyping reagents lack specificity and are not easily available, this monoclonal antibody could facilitate the management of patients with these disorders.

Antibodies, Monoclonal↗
PubMed1995

Surface recruitment but not activation of integrin alpha IIb beta 3 (GPIIb-IIIa) requires a functional actin cytoskeleton.

Binding of integrin alpha IIb beta 3 (glycoprotein [GP] IIb-IIIa) to soluble fibrinogen requires that the receptor undergo a conformational change (receptor activation), which occurs rapidly in agonist-stimulated platelets. Agonist stimulation of platelets also results in alpha IIb beta 3 recruitment from intracellular membranes (alpha-granules and open canalicular system) to the platelet surface. Once activated and accessible, the receptor can engage, a process that corresponds to the binding of the receptor to its soluble fibrinogen ligand, leading to intracellular signaling reactions and centripetal migration of bound receptor molecules. Because these processes occur concurrently with a marked reorganization of the actin cytoskeleton, we investigated the role of actin in fibrinogen receptor activation and surface recruitment. We used a flow cytometric assay to directly quantitate the binding of alpha IIb beta 3 to fluorescently labeled fibrinogen on the platelet surface. Cytochalasin D, which inhibits elongation of actin filaments, was used to prevent the actin response to platelet agonists. Despite its ability to inhibit the actin response and alpha IIb beta 3 binding to the actin cytoskeleton, cytochalasin D did not alter the agonist-induced intramolecular changes resulting in increased affinity of alpha IIb beta 3 for soluble fibrinogen and therefore did not inhibit ADP-induced aggregation. Thus, disruption of the actin network with cytochalasin D had no effect on the dissociation constant of the complex between activated alpha IIb beta 3 and fibrinogen (Kd = 0.26 to 0.28 mumol/L). However, cytochalasin D suppressed the recruitment of cryptic alpha IIb beta 3 molecules to the platelet surface.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗
PubMed1994

Rapid genotyping of the five major platelet alloantigens by reverse dot-blot hybridization.

Amino acid substitutions in platelet membrane glycoproteins result in alloantigens. Identifying these polymorphisms is important in alloimmune-mediated platelet disorders. Immunophenotyping platelet antigens can be limited by the unavailability of specific antisera. The goal of this work was to identify human platelet antigen genotypes in individuals using a technique that would circumvent the limitations of immunophenotyping and be clinically applicable. We have successfully applied the reverse dot-blot (RDB) technique to the genotyping of the five major human platelet alloantigen systems. Allele-specific oligonucleotides (ASOs) representing each allele of these alloantigens were covalently linked to a filter. Biotinylated oligonucleotides flanking the polymorphic sequences in genomic DNA were used to amplify genomic DNA by the polymerase chain reaction (PCR), and these products were hybridized to the filters containing the ASOs. Reactivity was detected with a chromogenic substrate. This nonradioactive methodology identifies all 15 possible genotypes in a well-defined control group of individuals and requires only two PCR reactions per patient sample. RDB analysis was used to successfully genotype women and family members with neonatal alloimmune thrombocytopenia and with posttransfusion purpura and to prenatally genotype the amniocytes from a fetus at risk for thrombocytopenia. The RDB methodology is specific, sensitive, and rapid and should enhance our ability to accurately diagnose disorders of alloimmune platelet destruction.

Alleles↗
PubMed1994

Isolation and characterization of a TATA-less promoter for the human beta 3 integrin gene.

Proper expression of the human platelet fibrinogen receptor is necessary for the maintenance of normal hemostasis. This receptor is formed by the heterodimer alpha IIb beta 3, a prototypic member of the integrin family of adhesive molecules. beta 3 is also expressed in other tissues with alpha v as the vitronectin receptor. It was not possible to study the basis for tissue-specific expression of this gene, because the beta 3 gene promoter had not been isolated previously. We have now isolated a 6.0-kb human genomic DNA fragment containing 2.0 kb of sequence 5' to the beta 3 ATG start codon. This clone also contains sequence encoding the signal peptide of the immature beta 3 protein and 3.0 kb of 3' intronic sequence. Primer extension and RNase protection studies of poly A+ RNA from a human erythroleukemia (HEL) cell line indicated a major transcription start site 30 bp upstream of the ATG start codon. In an orientation-dependent manner, a 584-bp fragment 5' to the start codon promotes expression of the chloramphenicol acetyl transferase (CAT) reporter gene in K562 cells. CAT expression from this beta 3 promoter is fivefold above expression from a "promoter-less" control CAT construct. This beta 3 promoter lacks TATA and CAAT cis-acting elements, but there are two Sp1 sites flanking the transcription start site. Other potential transcription factor binding sites are also identified. Phorbol esters (TPA), which increase beta 3 transcription in K562 cells, stimulated transcription from the 584-bp 5' beta 3 region. The isolation of this beta 3 promoter region should permit a more detailed analysis of its transcriptional regulation.

Amino Acid Sequence↗
PubMed1994

Platelet tyrosine kinases and fibrinogen receptor activation.

Platelet adhesion and aggregation during hemostasis and thrombosis are usually limited to sites where the integrity of the vessel wall is disrupted. The high concentration of platelet agonists within these sites represents a putative control mechanism for targeting platelet activation. Although much has been learned about the intracellular signaling systems controlling platelet activation, our understanding of the connection between signaling molecules and platelet aggregation remains limited. Tyrosine kinases are important signaling enzymes in cells and are abundant in platelets. Previous reports indicate that binding of glycoprotein IIb-IIIa (GPIIb-IIIa) to fibrinogen can induce the tyrosine phosphorylation of specific substrates. We show that, in turn, protein tyrosine kinase activity is necessary for agonist-induced activation of GPIIb-IIIa. Genistein and the tyrphostin AG-18 are two specific tyrosine kinase inhibitors, and the former has been shown to inhibit platelet aggregation. We use genistein and AG-18 in the present study to demonstrate that aggregation inhibition is due to suppression of GPIIb-IIIa activation. In contrast, genistin, an isoflavone compound related to genistein, and acetylsalicylic acid do not affect the tyrosine kinase-signaling pathway, nor do they inhibit GPIIb-IIIa activation induced by strong agonists. On identifying prominent tyrosine kinase substrates in activated platelets, we confirm that several substrates correspond to proteins associated with the cytoskeleton: the 85-kD subunit of phosphatidylinositol 3-kinase, the SH3-containing and actin-associating p85, pp60Src, and pp125FAK.(ABSTRACT TRUNCATED AT 250 WORDS)

Blood Platelets↗
PubMed1994

Quantitation of soluble fibrinogen binding to platelets by fluorescence-activated flow cytometry.

Soluble fibrinogen binding to agonist-stimulated blood platelets is the essential physiologic function of the glycoprotein IIb-IIIa (GPIIb-IIIa) receptor. We describe a method of quantifying this receptor-ligand interaction by using flow cytometry to detect the binding of fluorescein-labeled fibrinogen to activated platelets. Fibrinogen conjugated with fluorescein isothiocyanate (FITC-FGN) was structurally and functionally indistinguishable from native fibrinogen when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, thrombin clottability, and receptor affinity studies. Platelet samples, at a concentration of 2 x 10(7) ml, were incubated with FITC-FGN and activated with adenosine diphosphate (ADP) before cytometric acquisition of fluorescence and scatter data. ADP-induced binding of soluble FITC-FGN to platelet GPIIb-IIIa was specific, time dependent, and saturable. Cytometric analysis of FITC calibration beads allowed generation of standard curves relating bead fluorescence intensity to the number of fluorescein equivalents per bead. With this information, platelet fluorescence intensity was converted into the number of FITC-FGN molecules bound per platelet. Such quantitative analysis of fibrinogen binding yielded a dissociation constant of 2.48 +/- 0.5 x 10(-7) mol/L and a maximum fibrinogen binding capacity of 42, 124 +/- 5, 628 molecules per platelet (mean +/- SEM), which are comparable to published results with radioligand assays. The simplicity, sensitivity, and quantifiability of this method may make it a useful technique for basic and clinical research involving GPIIb-IIIa function.

Adenosine Diphosphate↗
PubMed1994

Inherited diseases of platelet glycoproteins: considerations for rapid molecular characterization.

The characterization of inherited diseases of platelets has provided valuable information about platelet physiology and platelet protein function. Genetic studies on patients with Glanzmann thrombasthenia, the Bernard-Soulier syndrome, and platelet-type von Willebrand disease have been confined to abnormalities of the GPIIb-IIIa and GPIb-IX receptor complexes. The primary molecular technique used in these analyses has been the polymerase chain reaction (PCR). The amplified PCR products are either directly sequenced, or used to screen for abnormal regions of the genes which are then sequenced. This review examines the known mutations in GPIIb-IIIa and GPIb-IX, focusing on those genetic issues which should dictate decisions regarding the approach to identifying molecular defects. The techniques for characterizing mutant alleles in Glanzmann thrombasthenia and Bernard-Soulier syndrome are described and a general strategy is offered. Because mutations resulting in reduced levels of transcripts can be missed when screening RNA, an argument is made for using genomic DNA as the primary material for mutation detection.

Alleles↗
PubMed1993

Single-strand conformation polymorphism analysis is a rapid and effective method for the identification of mutations and polymorphisms in the gene for glycoprotein IIIa.

Glanzmann thrombasthenia (GT) is the most common inherited disorder of platelets. Most of the molecular defects previously identified in GT have been caused by point (or other small) mutations in the genes for glycoprotein (GP) IIb or GPIIIa. We have used single-strand conformation polymorphism (SSCP) analysis to rapidly identify single-base changes in the GPIIIa gene. Using genomic DNA from normal individuals and patients with GT, each GPIIIa exon and a short stretch of flanking intronic sequence was amplified, heat-denatured, and separated in nondenaturing acrylamide gels. Only those fragments with an abnormal migration pattern were isolated and the nucleotide sequence determined. Using SSCP, we detected the polymorphism in the HPA-1 (P1A) system and all three known silent polymorphisms in the GPIIIa gene. Screening 14 GPIIIa exons from 5 patients with GT, one mutant allele was identified. The nucleotide sequence of the abnormal 240-bp SSCP fragment was determined and a G-->A substitution in the splice donor site of exon iv was identified. Analysis of platelet RNA resulting from this mutation showed two mRNA species: one contained a deletion of exon iv, whereas the other had a 27-bp addition to exon iv due to the use of a cryptic splice site in the downstream intron. Single-base substitutions are the most common mutation in GT and often result in abnormal mRNA splicing. SSCP is a rapid and sensitive technique for identifying mutations or polymorphisms in the GPIIIa gene.

Base Sequence↗
PubMed1993

Talin distribution and phosphorylation in thrombin-activated platelets.

We have previously demonstrated that the subcellular distribution of the adhesion plaque protein, talin, changes dramatically in human platelets in response to platelet activation (Beckerle et al., J. Cell Biol. 109, 3333-3346, 1989). Talin is uniformly distributed throughout the cytoplasm of resting platelets. However, when platelets are stimulated to become activated and adhesive, a significant amount of the talin population rapidly redistributes to a peripheral, submembranous location. In the present study we have examined talin phosphorylation and proteolytic cleavage as possible mechanisms by which talin's subcellular distribution could be regulated in platelets. We have found that thrombin activation of platelets leads to a fourfold increase in talin phosphorylation. Proteolytic cleavage of talin, however, is not detected in washed platelets activated with thrombin for as long as 30 minutes. Because talin moves to a submembranous location upon platelet activation and has been shown to interact with integrins in vitro, we also investigated whether the major platelet integrin, GPIIb-IIIa, is required for talin redistribution. Using Glanzmann thrombasthenic platelets, which are deficient in GPIIb-IIIa, we found that talin redistribution occurs even in the absence of GPIIb-IIIa. Collectively, our studies suggest that neither proteolytic cleavage of talin nor interactions between talin and GPIIb-IIIa is required for the regulated redistribution of talin in thrombin-activated platelets. Phosphorylation of talin in response to thrombin activation may, however, be one mechanism utilized by platelets to regulate talin distribution and function in human platelets.

Biological Transport↗
PubMed1993

Homologous recombination among three intragene Alu sequences causes an inversion-deletion resulting in the hereditary bleeding disorder Glanzmann thrombasthenia.

The crucial role of the human platelet fibrinogen receptor in maintaining normal hemostasis is best exemplified by the autosomal recessive bleeding disorder Glanzmann thrombasthenia (GT). The platelet fibrinogen receptor is a heterodimer composed of glycoproteins IIb (GPIIb) and IIIa (GPIIIa). Platelets from patients with GT have a quantitative or qualitative abnormality in GPIIb and GPIIIa and can neither bind fibrinogen nor aggregate. Very few genetic defects have been identified that cause this disorder. We describe a kindred with GT in which the affected individuals have a unique inversion-deletion mutation in the gene for GPIIIa. Patient platelets lacked both GPIIIa protein and mRNA. Southern blots of patient genomic DNA probed with an internal 1.0-kb GPIIIa cDNA suggested a large rearrangement of this gene but were normal when probed with small GPIIIa cDNA fragments that were outside the mutation. Cytogenetics and pulsed-field gel analysis of the GPIIIa gene were normal, making a translocation or a very large rearrangement unlikely. Additional Southern analyses suggested that the abnormality was not a small insertion. We constructed a patient genomic DNA library and isolated fragments containing the 5' and 3' breakpoints of the mutation. The nucleotide sequence from these genomic clones was determined and revealed that, relative to the normal gene, the mutant allele contained a 1-kb deletion immediately preceding a 15-kb inversion. The DNA breaks occurred in two inverted and one forward Alu sequence within the gene for GPIIIa and in the left, right, and left arms, respectively, of these sequences. There was a 5-bp repeat at the 3' terminus of the inversion.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1992

Demyelinating disease after neurologically complicated primary Epstein-Barr virus infection.

This report describes five patients who, following a neurologically complicated primary Epstein-Barr virus infection, developed progressive or relapsing neurologic deficits. The sequelae in four patients followed 4 to 12 years led to the diagnosis of multiple sclerosis (MS). The fifth patient presented with acute disseminated sclerosis and exhibits diffuse neurologic deficits that have persisted for 2 years. We suggest that the diagnosis of an unexplained acute neurologic or psychiatric syndrome should raise the question of a primary EBV etiology. A precisely timed serologic and hematologic study of the blood is imperative to capture the essential evidence. The data presented represent a clinical association between a neurologically complicated primary EBV infection and both chronic and acute demyelinating disease. The evidence does not justify a conclusion that EBV virus causes MS.

Adolescent↗
PubMed1992

Antibodies against Epstein-Barr nuclear antigen (EBNA) in multiple sclerosis CSF, and two pentapeptide sequence identities between EBNA and myelin basic protein.

The Epstein-Barr virus (EBV) causes infectious mononucleosis and is linked to several disparate malignancies. Prior studies on patients with multiple sclerosis (MS) showed that 100% are EBV-seropositive and that their blood contains higher antibody titers than those of controls to both transformation and lytic cycle antigens. We performed three different assays for antibodies in CSF to three major EBV antigens from patients with MS and controls. Among 93 patients with MS, 79 (85%) had CSF that reacted with a 70 kD protein, shown to be the nuclear antigen, EBNA-1, whereas only 11 (13%) of 81 EBV-seropositive controls reacted, p less than 0.001. The CSF of all 14 MS patients, unreactive on immunoblots, contained oligoclonal bands on agarose electrophoresis. Together, the two techniques exhibit 100% sensitivity in the confirmatory diagnosis of MS. We also performed amino acid searches of the Protein Identification Resource sequence database for protein homologies to EBNA. Two pentapeptide identities were found between EBNA-1 and myelin basic protein: QKRPS and PRHRD. None of more than 32,000 other proteins in the database contained both pentapeptides. In healthy EBV-seropositive persons, the EBV-specific, MHC-restricted T lymphocytes keep the EBV-containing B lymphocytes locked in the transformed state. However, in the host genetically susceptible to MS, the same population of lymphocytes might recognize and interact with either of the two identified pentapeptides, inadvertently damaging MBP.

Amino Acid Sequence↗
PubMed1991

Quantitative isolation of RNA from human platelets.

We have adapted the acid-guanidinium-phenol-chloroform extraction procedure of Chomczinsky and Sacchi to achieve efficient rapid recovery of total RNA from human platelets. Sufficient platelet RNA (20 micrograms of total RNA per 30 ml of whole blood) can be recovered from relatively small individual samples to perform Northern blot analysis on individual donors and detect the mRNAs for glycoproteins IIb1(GP IIb) and IIIa1(GP IIIa), 3.4 kb and 6.2 kb, respectively. Platelet GP IIb and GP IIIa mRNAs could also be reverse transcribed, and amplified in vitro by the polymerase chain reaction (PCR). Thus, our technique allows simultaneous Northern blotting and PCR, and therefore should be of great help to the characterization of inherited platelet disorders such as Glanzmann's thrombasthenia.

Blood Platelets↗
PubMed1990

Human platelets and megakaryocytes contain alternately spliced glycoprotein IIb mRNAs.

The earliest marker of the megakaryocyte lineage in human bone marrow is a heterodimeric complex of glycoproteins IIb (GPIIb) and IIIa (GPIIIa). GPIIb-IIIa is a member of the integrin family and serves as a receptor for fibrinogen and other adhesive glycoproteins on the surface of activated blood platelets. Mature platelet GPIIb is processed from a single precursor peptide into a disulfide-linked GPIIb alpha and GPIIb beta chain. To study possible mechanisms which could determine ligand specificity of the GPIIb-IIIa complex we investigated whether there are multiple forms of GPIIb mRNA by screening an unamplified lambda gt10 human erythroleukemia cell cDNA library. Four GPIIb clones were isolated and sequenced. Two contained a 102-nucleotide in-frame deletion corresponding to 34 amino acids in the extracellular domain of GPIIb beta. Polymerase chain reaction amplification of human erythroleukemia cell cDNA confirmed the presence of two different transcripts. Normal genomic DNA sequence was shown to contain consensus splice-donor and splice-acceptor sites indicating a 102-base pair exon that is spliced in or out. Using primers specific for either transcript, both forms of GPIIb mRNA were identified by the polymerase chain reaction in normal human platelets and megakaryocytes, but not in reticulocytes or two white blood cell lines. These data provide evidence for two forms of GPIIb in platelets and megakaryocytes and suggest a mechanism for variations in receptor ligand affinity or localization.

Amino Acid Sequence↗