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ABSTRACT: Op18 is a widely expressed, cell cycle-regulated, phosphoprotein involved in signal transduction of a variety of stimuli. In actively proliferating Jurkat T cells which express Op18 at high level, phorbol 12-myristate 13-acetate (PMA) treatment induces a rapid increase in the level of several Op18 phosphorylated forms. To determine phosphorylation sites involved in the PMA effect, the major Op18 phosphorylated forms were resolved in Jurkat T cells, before and after treatment with PMA, using preparative immobilized pH gradient-based two-dimensional polyacrylamide gel electrophoresis. Tryptic fragments of phosphorylated Op18 were analyzed by two-dimensional thin layer peptide mapping and were resolved by reverse-phase high performance liquid chromatography prior to analysis by matrix-assisted laser desorption ionization mass spectrometry. Phosphorylation sites were identified by further treatment of the proteolytic fragments with different enzymes and determination of the mass shifts by matrix-assisted laser desorption ionization mass spectrometry. Two major phosphorylation sites were identified. Low constitutive levels of phosphorylation at Ser25 and Ser38 in Op18a and Op18b was demonstrated. Treatment with PMA resulted in enhanced phosphorylation of Ser25 in Op18a and of both Ser25 and Ser38 in Op18b. Taken together with prior studies of Op18 phosphorylation, the data suggest that Op18 phosphorylation occurs at identical sites in different tissues and organisms.
Journal of Biological Chemistry 08/1993; 268(19):14269-77. · 4.77 Impact Factor
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ABSTRACT: We have developed a database of lymphoid polypeptides detected by two-dimensional polyacrylamide gel electrophoresis to aid in studies of leukemogenesis and of mutation affecting protein structure. In prior studies, we observed a 19-kDa phosphopolypeptide which was induced with proliferation in mature T cells and constitutively expressed in immature thymocytes. In this report we describe the identification of this polypeptide as the phosphorylated form of dUTPase (EC 3.6.1.23), following cDNA cloning of the gene, based on a partial amino acid sequence of the phosphopolypeptide. Studies of the expression and phosphorylation of dUTPase in human T cells indicate that accumulation and phosphorylation of dUTPase in mature T cells occur in a cell cycle-dependent manner. Interestingly, noncycling immature thymocytes express constitutively high levels of phosphorylated and unphosphorylated dUTPase. These results suggest an important role for dUTPase in immature thymocytes that is independent of proliferation.
Proceedings of the National Academy of Sciences 07/1993; 90(11):4991-5. · 9.68 Impact Factor
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ABSTRACT: The protein components of highly purified secretory granule membranes and the granule contents from rat exocrine pancreas were characterized by two-dimensional polyacrylamide gel electrophoresis, protein staining, lectin absorption, and Western blotting with anti-secretory protein antibodies. NH2-terminal amino acid sequence was obtained for a approximately 53-kDa glycoprotein denoted GP-3, present only in granule membrane preparations where it was resistant to washing with Na2CO3 and KBr. The sequence of this protein showed homology to pancreatic lipase but was distinct from the NH2-terminal sequence of a 50-kDa content protein presumed to be secretory lipase. Polymerase chain reaction amplification with degenerate oligonucleotide primers to GP-3 and secretory lipase gave partial length subclones that were used to isolate clones from a rat pancreas cDNA library. Dideoxy sequencing of full-length subclones of GP-3 revealed the predicted amino acid sequence for a mature protein of 452 amino acids with a potential N-linked glycosylation site and a deglycosylated molecular weight of 50,860. The GP-3 sequence possesses the serine esterase consensus sequence G-X-S-X-G centered around Ser154 and the catalytic state triad Asp178-His265-Ser154 characteristic of pancreatic lipases. Northern blot analysis of various rat tissues showed GP-3 expression solely in pancreas. Comparison of GP-3 nucleotide and amino acid sequence, along with pancreatic lipases of various species including rat, shows extensive homologies to both proteins and reveals an underlying diversity in the pancreatic lipase family. Close homology is observed between GP-3 and a lipase molecule previously isolated from mouse cytotoxic T cells.
Journal of Biological Chemistry 06/1993; 268(14):10303-11. · 4.77 Impact Factor
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ABSTRACT: Insulin was isolated from the pancreas of the American eel, Anguilla rostrata, and its primary structure was established as (Formula: see text). Eel insulin contains unusual substitutions at B-21, B-22, and B-26 in the putative receptor-binding region of the molecule compared with other mammalian and fish insulins. The A-chain of insulin from the European eel contains an asparagine rather than a serine residue at position A-12. Similarly, amino acid composition data indicate the B-chain of insulin from the European eel is appreciably different from that from the American eel. The primary structure of glucagon-like peptide (GLP) from the American eel is identical to that from the European eel, Anguilla anguilla. The primary structure of the peptide was established as (Formula: see text). Fast-atom bombardment mass spectrometry demonstrated that the COOH-terminal arginyl residue is alpha-amidated. The strong evolutionary pressure to conserve the structure of GLP provides further support for the assertion that the peptide plays an important regulatory role in teleost fish.
General and Comparative Endocrinology 05/1991; 82(1):23-32. · 3.27 Impact Factor
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ABSTRACT: Immunoreactivity of antisera directed against human neuropeptide Y (NPY), anglerfish polypeptide YG (aPY), bovine pancreatic polypeptide (bPP), salmon pancreatic polypeptide (sPP), porcine peptide tyrosine tyrosine (PYY), and salmon glucagon-like peptide (GLP) was investigated in the endocrine pancreas and anterior intestine of adult lampreys, Petromyzon marinus, by immunohistochemical analysis. There was no immunoreactivity to anti-sPP and anti-bPP in any tissue and anti-GLP immunostaining was only present in the anterior intestine. The immunoreactivity to antisera raised against NPY, aPY, and PYY was colocalized within the same small number of cells in the caudal and cranial pancreas of juveniles and the caudal pancreas of upstream migrant adults. These antibodies did not immunostain B- or D-cells and thus, NPY, aPY, and PYY were likely localized in a third cell type (3a) in the lamprey pancreas. Immunostaining of a few cells with only anti-aPY suggested the possibility of a fourth cell type (3b). Immunoreactivity was similar in the cranial and caudal pancreas of male upstream migrants; however, in the female cranial pancreas, a few cells demonstrated intense immunoreaction to anti-aPY, while weaker immunostaining with this antiserum was observed in B-cells. In the intestine of juvenile and upstream migrant lampreys, positive immunostaining to GLP, NPY, aPY, and PYY antibodies was colocalized within the same cell. We believe that this cell may contain PYY/glucagon family peptides. Other intestinal cells immunostained with either GLP or somatostatin-34 antiserum.
General and Comparative Endocrinology 02/1991; 81(1):51-63. · 3.27 Impact Factor
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ABSTRACT: A new procedure to selectively identify disulfide-containing peptides in extracts of biological tissues is described. Disulfide-containing peptides are detected by their UV absorbance and electrochemical (EC) activity after chromatographic separation, and subsequently identified by fast atom bombardment mass spectrometry (FABMS). This combination of fractionation by HPLC and selective detection is attractive because it is rapid, highly specific for disulfide-containing peptides, and applicable to all disulfide-containing peptides that may be present in complex biological mixtures. Useful procedures for applying the method are demonstrated with tissue extracts from bovine pituitary and catfish pancreas. In addition to finding the expected disulfide-containing peptides, evidence for two forms of catfish insulin are presented. The merits of this and other methods used to detect peptides in similar tissue extracts are discussed.
Journal of Protein Chemistry 05/1990; 9(2):151-7.
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ABSTRACT: A full-length cDNA encoding porcine heart aconitase was derived from lambda gt10 recombinant clones and by amplification of the 5' end of the mRNA. The 2700-base pair (bp) cDNA contains a 29-bp 5' untranslated region, a 2343-bp coding segment, and a 327-bp 3' untranslated region. The porcine heart enzyme is synthesized as a precursor containing a mitochondrial targeting sequence of 27 amino acid residues which is cleaved to yield a mature enzyme of 754 amino acids, Mr = 82,754, having a blocked amino terminus. The NH2-terminal pyroglutamyl residue of the mature enzyme was identified by fast atom bombardment mass spectrometry and sequence analyses of an NH2-terminal peptide. Mature porcine heart aconitase contains 12 cysteine residues. Cysteines 358, 421, and 424 are ligands to the Fe-S cluster in the inactive [3Fe-4S] (Robbins, A. H., and Stout, C. D. (1989) Proteins 5, 289-312) and active [4Fe-4S] (Robbins, A. H., and Stout, C. D. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 3639-3643) forms. An alignment of the derived porcine heart sequence with 8 cysteine-containing tryptic peptides from bovine heart aconitase (Plant, D. W., and Howard, J. B. (1988) J. Biol. Chem. 263, 8184-8189; Plank, D. W., Kennedy, M. C., Beinert, H., and Howard, J. B. (1989) J. Biol. Chem. 264, 20385-20393) shows that 198 of 202 amino acids are conserved and suggests that the two enzymes are virtually identical.
Journal of Biological Chemistry 03/1990; 265(5):2814-21. · 4.77 Impact Factor
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ABSTRACT: D-Xylose is a major component of the carbohydrates derived from agricultural residues and forest products. Among more than two hundred known xylose-utilizing yeasts, only a few species are known to be able to ferment xylose anaerobically. Candida shehatae is one of such xylose-fermenting yeasts. Xylose reductase (E.C. 1.1.1.21) is a key enzyme responsible for xylose metabolism in xylose-utilizing as well as xylose-fermenting yeasts. In this paper, we report the development of a convenient and reliable procedure for the purification of xylose reductase from C. shehatae to near homogeneity. The amino acid composition and N-terminal sequence of the enzyme have also been analyzed. C. shehatae seems to contain only a single xylose reductase, but the enzyme has a dual coenzyme specificity for both NADPH and NADH. The enzyme is remarkably stable at room temperature and 4 degrees C.
Enzyme and Microbial Technology 02/1990; 12(1):33-9. · 2.37 Impact Factor
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International journal of peptide and protein research 09/1989; 34(2):158-60.
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ABSTRACT: Results from a previous report demonstrate that more than one molecular form of neuropeptide Y-like peptide may be present in the islet organ of the anglerfish (Lophius americanus). Most of the neuropeptide Y-like immunoreactive material was anglerfish peptide YG, which is expressed in a subset of islet cells, whereas an additional neuropeptide Y-like peptide(s) was localized in islet nerves. To learn more about the neuropeptide Y-like peptides in islet nerves, we have employed immunohistochemical and biochemical methods to compare peptides found in anglerfish islets and brain. Using antisera that selectively react with either mammalian forms of neuropeptide Y or with anglerfish peptide YG, subsets of neurons were found in the brain that labelled with only one or the other of the antisera. In separate sections, other neurons that were labelled with either antiserum exhibited similar morphologies. Peptides from brains and islets were subjected to gel filtration and reverse-phase high performance liquid chromatography. Radioimmunoassays employing either the neuropeptide Y or peptide YG antisera were used to examine chromatographic eluates. Immunoreactive peptides having retention times of human neuropeptide Y and porcine neuropeptide Y were identified in extracts of both brain and islets. This indicates that peptides structurally similar to both of these peptides from the neuropeptide Y-pancreatic polypeptide family are expressed in neurons of anglerfish brain and nerve fibers of anglerfish islets. The predominant form of neuropeptide Y-like peptide in islets was anglerfish peptide YG. Neuropeptide Y-immunoreactive peptides from islet extracts that had chromatographic retention times identical to human neuropeptide Y and porcine neuropeptide Y were present in much smaller quantities.(ABSTRACT TRUNCATED AT 250 WORDS)
Cell and Tissue Research 09/1989; 257(2):303-11. · 3.11 Impact Factor
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ABSTRACT: The biotin-binding site of acetyl-CoA carboxylase from rat was characterized as to its amino acid sequence and relative position in the enzyme molecule. Biotin binds to the lysyl residue in the tetrapeptide Val-Met-Lys-Met; this tetrapeptide is located in close proximity to the NH2 terminus. In all other biotin-containing enzymes, the conserved tetrapeptide Ala-Met-Lys-Met is the counterpart to that of rat acetyl-CoA carboxylase; and the lysyl residue is 35 residues from the COOH terminus. To examine the significance of these unusual features of the biotinylation site of animal acetyl-CoA carboxylase, cDNA fragments were expressed in a bacterial system and the effects of specific site-directed mutagenesis were examined. Replacement of Val by Ala in the conserved tetrapeptide abolished biotinylation of the expressed protein. However, introduction of a termination codon at residue 36, in such a way that the distance between the lysine on which biotin binds and the COOH-terminal amino acid was 35 residues and the penultimate amino acid was the hydrophobic residue leucine, increased the efficiency of biotinylation, provided a substantial portion of the NH2-terminal peptide was removed.
European Journal of Biochemistry 07/1989; 182(2):239-45. · 3.58 Impact Factor
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ABSTRACT: The primary structure of an insulin isolated from the pancreas of the holocephalan fish, Hydrolagus colliei (Pacific ratfish), has been established as A-chain: GIVEQCCHNTCSLANLEGYCN B-chain: VPTQRLCGSHLVDALYFVCGERGFFYSPKPIRELEPLL. Three further molecular forms of insulin were also isolated and shown to have the same A-chain but truncated B-chains of 31-, 36-, and 37-amino acid residues. It is proposed that all four insulins arise from a single proinsulin by proteolytic cleavages at different sites within the C-peptide region. The insulin with 38 amino acids in the B-chain was equipotent with human insulin in inhibiting the binding of radiolabelled human insulin to rat fat cells but the maximum effect of ratfish insulin upon the transport of 3-O-methylglucose into the cells was only 65% of the maximum effect of human insulin. Two molecular forms of glucagon-like peptide were isolated from the ratfish pancreas. The primary structure of the more abundant peptide was established as HADGIYTSDVASLTDYLKSKRFVESLSNYNRKQND. The primary structure of the second peptide was the same except that it was extended from the C-terminus by the sequence RRM. It is probable, therefore, that both glucagon-like peptides also arise from a single proglucagon by different pathways of post-translational processing.
General and Comparative Endocrinology 02/1989; 73(1):136-46. · 3.27 Impact Factor
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ABSTRACT: The anglerfish endocrine pancreas expresses two different genes for preproglucagon. The regions of the two proglucagons that correspond to glucagon have different sequences, as do the two glucagon-like peptides (GLPs). The products derived from processing the more abundant proglucagon-II have recently been determined. However, it was not known whether proglucagon-I was processed to similar products. The two major biologically active products of preproglucagon-I processing (glucagon-I and GLP-I) have now been purified to homogeneity. Their structures were determined using automated gas phase Edman degradation, tryptic mapping, and fast atom bombardment mass spectrometry. The preproglucagon-I-processing sites were identified. Glucagon-I represents residues 53-81, and GLP-I corresponds to preproglucagon-I-(91-124) (numbering from the initiator Met).
Endocrinology 01/1989; 123(6):2639-45. · 4.46 Impact Factor
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ABSTRACT: Insulin has been isolated from the pancreas of the holocephalan fish, Chimaera monstrosa (rabbit fish), and characterized by automated Edman degradation and fast atom bombardment mass spectrometry. The primary structure of rabbit fish insulin was identical to that of insulin from the holocephalan fish, Hydrolagus colliei (Pacific ratfish), and contained 21 residues in the A-chain and 38 residues in the B-chain. The amino acid compositions of both rabbit fish and ratfish insulins demonstrated a value consistently lower than that expected for the leucine content of the peptides. It is suggested, therefore, that the insulins were probably isolated as a mixture of the intact peptides and components lacking the C-terminal leucine residue in the B-chain.
General and Comparative Endocrinology 11/1988; 72(1):154-60. · 3.27 Impact Factor
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ABSTRACT: Californium-252 plasma desorption mass spectrometry (252Cf PDMS) of a crude, desalted, extract of piscine endocrine pancreas provided mass information for the major biologically active peptide hormones present in this tissue. An extraction procedure compatible with 252Cf PDMS analysis was developed. In extracts of catfish pancreas, strong molecular ions were identified in the positive mode for somatostatin-14 (1638 amu), O-glycosylated somatostatin-22 (2944 amu), glucagon (3512 amu), glucagon-like peptide (3785 amu), insulin (ca. 5550 amu), and other prohormone-derived peptides. Both protonated species and sodium adducts were apparent in the mass spectrum. A number of other molecular ions were observed including somatostatin-26, 1-10 (1014 amu) and the entire portion of prosomatostatin-22 remaining after removal of somatostatin-22 (6465 amu). The data obtained by this method also resulted in the identification of the third major product of proglucagon processing in catfish pancreas, glicentin-related polypeptide. Subtractive Edman degradation analyzed by 252Cf PDMS was also used to confirm a mass assignment.
Analytical Biochemistry 11/1988; 174(1):23-31. · 3.00 Impact Factor
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ABSTRACT: Three major forms of somatostatin were isolated from pancreas of the lamprey (Petromyzon marinus). One of the two major forms is a 14-residue somatostatin (SS-14) having the sequence AGCKNFFWKTFSSC. The homologous substitution Ser for Thr in position 12 is the first example of SS-14 from vertebrate preprosomatostatin gene I having a divergent sequence. The longest form is 37 residues in length (SS-37) and has the sequence ALRAAAVAGSPQQLLPLGQRERKAGCKNFFWKTFSSC. A 34-residue form (SS-34) identical in sequence but truncated at a single Arg residue at position 3 of SS-37 was also isolated. The yields of the three forms were SS-37 (0.43 nmol/g), SS-34 (134 nmol/g), and SS-14 (51.5 nmol/g). The identification of this nested series of somatostatins suggests that prosomatostatin processing in lamprey more closely resembles that observed for procholecystokinin than that of mammalian or other piscine prosomatostatins. Somatostatin-producing cells in the lamprey pancreas were identified by immunostaining using antiserum against SS-34 and anti-serum against mammalian SS-14.
Journal of Biological Chemistry 11/1988; 263(30):15809-14. · 4.77 Impact Factor
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ABSTRACT: The nucleotide sequence of soybean lipoxygenase-2 cDNA has been determined, and the complete amino acid sequence of the enzyme has been deduced. Limited direct amino acid sequence data for lipoxygenase-2 protein support this assignment and exclude mRNA representing lipoxygenase-1 and -3. Lipoxygenase 2 has a molecular weight of 97,036 and contains 865 amino acid residues, in contrast to the isozymes, lipoxygenase-1 and -3, which are known to contain 838 and 859 amino acid residues, respectively. Despite significant differences in behavior between these three isozymes, the amino acid sequences of lipoxygenase-1 and -3 are 81 and 74% identical to lipoxygenase-2, respectively. A region of 40 amino acid residues containing a cluster of six histidines and two tyrosines, which is highly conserved in all three isozymes, is discussed as a possible iron-binding region.
Journal of Biological Chemistry 06/1988; 263(14):6816-21. · 4.77 Impact Factor
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ABSTRACT: Insulin has been purified to homogeneity from the caudal and cranial pancreas of the adult sea lamprey (Petromyzon marinus). The final yield was 18.6 nmol/g (127.8 micrograms/g). The structures of both A- and B-chains have been determined using amino acid analyses, gas-phase sequence analyses, and proteolytic mapping by fast atom bombardment-mass spectrometry. The sequence of the A-chain was found to be GIVEQCCHRKCSIYDMENYCN. The sequence of the B-chain, extended at the amino terminus, was determined to be SALT-GAGGTHLCGSHLVEALYVVCGDRGFFYTPSKT. Lamprey insulin retains the common features of vertebrate insulins. Sea lamprey insulin has no more homology to hagfish (Myxine glutinosa) insulin than it has to the teleost fish or to mammalian insulins.
General and Comparative Endocrinology 02/1988; 69(1):46-55. · 3.27 Impact Factor
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ABSTRACT: The products and an intermediate of preprosomatostatin-II processing in the anglerfish islet were purified and subjected to structural analysis. The peptides isolated identify the site of signal cleavage (between Ser-24 and Gln-25). The prohormone is further processed at Arg-97 and, to a lesser extent, at the two adjacent basic amino acid residues Lys-61 and Arg-62. A 28-residue somatostatin is also generated which can be hydroxylated at Lys-23. A proteolytic processing site which would form the 14-residue somatostatin does not appear to be used to a significant degree. Fast atom bombardment mass spectrometry (FABMS) was used to demonstrate that the amino-terminal residues of peptides 25-60, and 25-90 are pyroglutamic acid, a modification which precludes Edman degradation of these peptides. Analysis of the peptides and tryptic peptide maps by FABMS allowed confirmation of the sites of prohormone conversion and indicated that terminal basic residues were removed during processing. Three amino acid residues were also found to differ from the amino acid sequence deduced from the cDNA and were localized to specific regions by FABMS analysis. Residues found to differ from the cDNA (cDNA in parentheses) were: Asp-77 (Thr), Val-78 (Phe), and Gly-90 (Glu). Mass assignments were confirmed by running a single cycle of Edman degradation prior to FABMS. The peptides noted above were also examined by Edman sequence analysis. The sequence of a cDNA clone to preprosomatostatin-II was re-examined in light of the observed differences at the protein level. This study emphasizes the utility of FABMS in prohormone processing studies and in identification of post-translational processing events.
Journal of Biological Chemistry 10/1987; 262(26):12692-9. · 4.77 Impact Factor
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ABSTRACT: Major products and an intermediate in the proteolytic processing pathway of preprosomatostatin I from anglerfish (Lophius americanus) were purified and characterized. Proteolytic mapping by fast atom bombardment mass spectrometry was used to rapidly locate regions of the peptides whose masses deviated from those deduced from the cDNA sequence. Amino acid analysis and partial Edman sequencing were also used to confirm the structures. The protein structural data indicate a Glu for Gly substitution at position 83 of preprosomatostatin I (aPPSS-I, numbering from the initiator Met) relative to the cDNA sequence. Two of the peptides isolated, aPPSS-I (26-52) (7.5 nmol X g-1) and aPPSS-I (26-92) (49.5 nmol X g-1), define signal cleavage as occurring between Cys-25 and Ser-26. A partial sequence was obtained from fragment ions in the mass spectrum of a peptide corresponding to aPPSS-I (94-105) (58 nmol X g-1). The 14-residue somatostatin [SS-14 corresponding to aPPSS-I (108-121)] has previously been isolated [Noe, B. D., Spiess, J., Rivier, J. E., & Vale, W. (1979) Endocrinology (Baltimore) 105, 1410-1415]. Taken together, these peptides suggest a pathway for prosomatostatin I processing in which the residues corresponding to SS-14 and the immediately preceding 14 residues are cleaved from the prohormone via endoproteolysis (order of cleavage not determined). The fragment aPPSS-I (94-105) was isolated in lower yield than SS-14 and may represent a secondary site of cleavage. Subsequent cleavage at arginine-53 results in the minor peptide aPPSS-I (26-52).(ABSTRACT TRUNCATED AT 250 WORDS)
Biochemistry 08/1987; 26(15):4853-61. · 3.42 Impact Factor
