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J M Becker

Publications and source records attributed to J M Becker.

At least 37 records · Page 2Linked to original sources
PubMed2000

Position 13 analogs of the tridecapeptide mating pheromone from Saccharomyces cerevisiae: design of an iodinatable ligand for receptor binding.

Analogs of the alpha-factor tridecapeptide mating pheromone (WHWLQLKPGQPMY) from Saccharomyces cerevisiae in which Tyr13 was replaced with Phe, p-F-Phe, m-F-Phe, p-NO2-Phe, p-NH2-Phe or Ser were synthesized and purified to >99% homogeneity. These analogs were bioassayed using a growth arrest assay and a gene induction assay and evaluated for their ability to compete with binding of tritiated alpha-factor to its receptor Ste2p. The results showed that the phenolic OH of Tyr13 is not required for either biological activity or receptor recognition. Analogs containing fluorine, amino, nitro or a hydrogen in place of OH had 80-120% of the biological activity of the parent pheromone in the gene induction assay and had receptor affinities from nearly equal to 6-fold lower than that of alpha-factor. In contrast, substitution of Ser or Ala at position 13 resulted in a >100-fold decrease in receptor affinity suggesting that the aromatic ring is involved in binding to the receptor. The lack of a strict requirement for Tyr13 allowed the design of several multiple replacement analogs in which Phe or p-F-Phe were substituted at position 13 and Tyr was placed in other positions of the peptide. These analogs could then be iodinated and used in the development of a highly sensitive receptor-binding assay. One potential receptor ligand [Tyr(125I)1,Nle12, Phe13] alpha-factor exhibited saturable binding with a KD of 81 nM and was competed by alpha-factor for binding in a whole-cell assay. Thus a new family of radioactive ligands for the alpha-factor receptor has been revealed. These ligands should be extremely useful in defining active site residues during mutagenesis and cross-linking studies.

Binding, Competitive↗
PubMed2000

NK-1 antagonist reduces colonic inflammation and oxidative stress in dextran sulfate-induced colitis in rats.

Although substance P (SP) has been implicated as a mediator of neurogenic inflammation in the small intestine, little information is available regarding the role of SP in the pathogenesis of chronic ulcerative colitis. In this study, our aim was to investigate whether the intraperitoneal administration of a nonpeptide neurokinin-1 (NK-1) antagonist, CP-96345, which antagonizes the binding of SP to its NK-1 receptor, results in the attenuation of colonic inflammation induced in rats by 5% dextran sodium sulfate (DSS) in drinking water for 10 days compared with an inactive enantiomer, CP-96344. Disease activity was assessed daily for 10 days, after which colonic tissue damage was scored and myeloperoxidase activity and colon and urinary 8-isoprostanes were measured. Animals receiving DSS exhibited marked physical signs of colitis by day 5 compared with controls. Chronic administration of the NK-1 antagonist significantly reduced the disease activity index, mucosal myeloperoxidase activity, colonic tissue damage score, and mucosal and urinary levels of 8-isoprostanes compared with inactive enantiomer- or vehicle-injected (saline) animals receiving DSS alone. These data indicate that the administration of an NK-1 antagonist can attenuate colonic inflammation and oxidative stress and suggest a novel therapeutic approach in the treatment of chronic ulcerative colitis.

Animals↗
PubMed1999

Oral versus intravenous corticosteroids in children hospitalized with asthma.

BACKGROUND: Previous studies have demonstrated that in the emergency treatment of an asthma exacerbation, corticosteroids used in conjunction with beta-agonists result in lower hospitalization rates for children and adults. Furthermore, orally administered corticosteroids have been found to be effective in the treatment of outpatients with asthma. However, similar data in inpatients is lacking. OBJECTIVE: The purpose of this study was to determine the efficacy of oral prednisone versus intravenous methylprednisolone in equivalent doses for the treatment of an acute asthma exacerbation in hospitalized children. METHODS: We conducted a randomized, double-blind, double-placebo study comparing oral prednisone at 2 mg/kg/dose (maximum 120 mg/dose) twice daily versus intravenous methylprednisolone at 1 mg/kg/dose (maximum 60 mg/dose) four times daily in a group of patients 2 through 18 years of age hospitalized for an acute asthma exacerbation. All patients were assessed by a clinical asthma score 3 times a day. The main study outcome was length of hospitalization; total length of stay and time elapsed before beta-agonists could be administered at 6-hour intervals. Duration of supplemental oxygen administration and peak flow measurements were secondary outcome measures. RESULTS: Sixty-six patients were evaluated. Children in the prednisone group had a mean length of stay of 70 hours compared with 78 hours for the methylprednisolone group (P =.52). Children in the prednisone group were successfully weaned to beta-agonists in 6-hour intervals after 59 hours compared with 68 hours for the methylprednisolone group (P =.47). Patients receiving prednisone required supplemental oxygen for 30 hours compared with 52 hours for the methylprednisolone group (P =.04). CONCLUSION: There was no difference in length of hospital stay between asthmatic patients receiving oral prednisone and those receiving intravenous methylprednisolone. Because hospitalization charges are approximately 10 times greater for intravenous methylprednisolone compared with oral prednisone, the use of oral prednisone to treat inpatients with acute asthma would result in substantial savings.

Administration, Oral↗
PubMed1999

Surgical therapy for ulcerative colitis and Crohn's disease.

Over the past 2 decades there has been considerable progress in the surgical management of inflammatory bowel disease. Crohn's disease is a chronic, nonspecific inflammatory disease of the gastrointestinal tract of unknown cause. It involves mainly the ileum, colon, and rectum, most often producing symptoms of obstruction or localized perforation with fistula. Although surgical treatment is palliative, operative excision in combination with strictureplasty, where appropriate, provides effective symptomatic relief and reasonable long-term benefit. Chronic ulcerative colitis is a diffuse inflammatory disease of the mucosal lining of the colon and rectum. Total removal of the colon and rectum provides a complete cure. Newer surgical alternatives, developed over the last 2 decades, have eliminated the need for a permanent ileostomy following definitive resection of the involved colon and rectum.

Anal Canal↗
PubMed1999

Long-distance rotational echo double resonance measurements for the determination of secondary structure and conformational heterogeneity in peptides.

The utility of rotational echo double resonance (REDOR) NMR spectroscopy for determining the conformations of linear peptides has been examined critically using a series of crystalline and amorphous samples. The focus of the present work was the evaluation of long-distance (> 5 A) interactions using 13C-15N dephasing. Detailed studies of specifically labeled melanostatin and synthetic analogs of the alpha-factor yeast mating hormone show that nitrogen-dephased, carbon-observe REDOR measurements are reliable for distances up to 6.0 A, and that dipolar interactions can be detected for distances up to 7 A. By contrast, nitrogen-observe REDOR gives reliable results only for distances shorter than 5.0 A. To measure distances accurately, REDOR data must be corrected for the effects of natural-abundance spins. These corrections are particularly important for measuring long distances, which are of the greatest value for determining peptide secondary structure. We have developed a spherical shell model for calculating the effect of these background spins. The REDOR studies also indicate that in a lyophilized powder, the tridecapeptide alpha-factor mating pheromone from Saccharomyces cerevisiae (WHWLQLKPGQPMY) probably exists as a distribution of different turn structures around the KPGQ region. This finding revises previous solid-state NMR studies on this peptide, which concluded alpha-factor assumes a distorted type-I beta-turn in the Pro-Gly central region of the molecule [J.R. Garbow, M. Breslav, O. Antohi, F. Naider, Biochemistry, 33 (1994) 10094].

Algorithms↗
PubMed1999

WdChs4p, a homolog of chitin synthase 3 in Saccharomyces cerevisiae, alone cannot support growth of Wangiella (Exophiala) dermatitidis at the temperature of infection.

By using improved transformation methods for Wangiella dermatitidis, and a cloned fragment of its chitin synthase 4 structural gene (WdCHS4) as a marking sequence, the full-length gene was rescued from the genome of this human pathogenic fungus. The encoded chitin synthase product (WdChs4p) showed high homology with Chs3p of Saccharomyces cerevisiae and other class IV chitin synthases, and Northern blotting showed that WdCHS4 was expressed at constitutive levels under all conditions tested. Reduced chitin content, abnormal yeast clumpiness and budding kinetics, and increased melanin secretion resulted from the disruption of WdCHS4 suggesting that WdChs4p influences cell wall structure, cellular reproduction, and melanin deposition, respectively. However, no significant loss of virulence was detected when the wdchs4Delta strain was tested in an acute mouse model. Using a wdchs1Delta wdchs2Delta wdchs3Delta triple mutant of W. dermatitidis, which grew poorly but adequately at 25 degrees C, we assayed WdChs4p activity in the absence of activities contributed by its three other WdChs proteins. Maximal activity required trypsin activation, suggesting a zymogenic nature. The activity also had a pH optimum of 7.5, was most stimulated by Mg(2+), and was more inhibited by polyoxin D than by nikkomycin Z. Although the WdChs4p activity had a broad temperature optimum between 30 to 45 degrees C in vitro, this activity alone did not support the growth of the wdchs1Delta wdchs2Delta wdchs3Delta triple mutant at 37 degrees C, a temperature commensurate with infection.

Amino Acid Sequence↗
PubMed1998

Mutations affecting ligand specificity of the G-protein-coupled receptor for the Saccharomyces cerevisiae tridecapeptide pheromone.

Random mutations were generated in the G-protein-coupled receptor (Ste2p) for the tridecapeptide pheromone (alpha-factor) of Saccharomyces cerevisiae. These mutants were screened for variants that responded to antagonists. Because multiple mutations were detected in each mutant receptor recovered from the screen, site-directed mutagenesis was used to create single-site mutant receptors. Three receptors containing mutations F55V, S219P, and S259P were analyzed for their biological responses to various alpha-factor analogs and for their ligand binding profiles. Cells expressing each of the mutant receptors responded to alpha-factor as well as or better than wild-type cells in a growth arrest assay. In contrast, the binding of alpha-factor to the F55V and S219P mutant receptors was at least 10-fold reduced in comparison to wild-type receptor indicating a complex non-linear correlation between binding affinity and biological activity. Cells expressing mutant receptors responded to some normally inactive analogs in biological assays, despite the fact that these analogs had a low affinity for Ste2p. The analysis of these mutant receptors confirms previous findings that the first and sixth transmembrane regions of Ste2p are important for ligand interaction, ligand specificity, and/or receptor activation to initiate the signal transduction pathway. Changes in binding affinity of pheromone analogs to wild-type and mutant receptors indicate that residue 55 of Ste2p is involved with both ligand binding and signal transduction.

Binding Sites↗
PubMed1998

Synthesis, biological activity, and conformational analysis of peptidomimetic analogues of the Saccharomyces cerevisiae alpha-factor tridecapeptide.

Biochemical and biophysical investigations on the Saccharomyces cerevisiae alpha-factor indicate that this tridecapeptide mating pheromone (WHWLQLKPGQPMY) might adopt a type II beta-turn in the center of the peptide when it binds to its G protein-coupled receptor. To test this hypothesis we synthesized analogues of alpha-factor incorporating a (R or S)-gamma-lactam conformational constraint [3-(R or S)-amino-2-oxo-1-pyrrolidineacetamido] in place of the Pro-Gly at residues 8 and 9 of the peptide and tested their biological activities and receptor binding. Analogues were purified to >99% homogeneity as evidenced by high-performance liquid chromatography and capillary electrophoresis and characterized by amino acid analysis, mass spectrometry, and nuclear magnetic resonance (NMR) spectroscopy. The restricted alpha-factor analogue WHWLQLK[(R)-gamma-lactam]QP[Nle]Y was more active than its lactam-containing diastereomeric homologue WHWLQLK[(S)-gamma-lactam]QP[Nle]Y and about equally active with the [Nle12]-alpha-factor in growth arrest and FUS1-lacZ gene induction assays. Both lactam analogues competed with tritiated [Nle12]-alpha-factor for binding to the alpha-factor receptor (Ste2p) with the (R)-gamma-lactam-containing peptide having 7-fold higher affinity than the (S)-gamma-lactam-containing homologue. Two-dimensional NMR spectroscopy and modeling analysis gave evidence that the (R)-gamma-analogue is a flexible peptide that assumes a transient gamma-turn structure around the lactam moiety. The results represent the first example of an alpha-factor analogue containing a peptidomimetic constraint that is as active as the native pheromone. The correlation between activity and structure provides further evidence that the biologically active conformation of the molecule contains a turn in the middle of the pheromone. This study provides new insights into the structural basis of alpha-factor activity and adds to the repertoire of conformationally biasing constraints that can be used to maintain and even enhance biological activity in peptide hormones.

Amides↗
PubMed1998

Linkage of adhesion, filamentous growth, and virulence in Candida albicans to a single gene, INT1.

Adhesion and the ability to form filaments are thought to contribute to the pathogenicity of Candida albicans, the leading cause of fungal disease in immunocompromised patients. Int1p is a C. albicans surface protein with limited similarity to vertebrate integrins. INT1 expression in Saccharomyces cerevisiae was sufficient to direct the adhesion of this normally nonadherent yeast to human epithelial cells. Furthermore, disruption of INT1 in C. albicans suppressed hyphal growth, adhesion to epithelial cells, and virulence in mice. Thus, INT1 links adhesion, filamentous growth, and pathogenicity in C. albicans and Int1p may be an attractive target for the development of antifungal therapies.

Animals↗
PubMed1998

Conformational analysis of cyclic analogues of the Saccharomyces cerevisiae alpha-factor pheromone.

Analogues of the alpha-factor mating pheromone (WHWLQLKPGQPMY) from Saccharomyces cerevisiae in which the side chains of residues 7 and 10 were joined by lactam bonds were studied by nmr and molecular modeling. These investigations were carried out to discern the effect of lactam ring size on conformation and to ascertain whether the side chain i to i + 3 cyclized tetramers [H. R. Marepalli et al. (1996) Journal of the American Chemical Society, Vol. 118, pp. 6531-6539] can be considered as conformation-constraining building blocks when introduced into a long peptide chain. Nuclear Overhauser effect constraints, temperature coefficients, and backbone torsional angles were derived from 1H-nmr spectra measured in DMSO-d6. Modeling studies using the above constraints indicate that the lactam regions of the tridecapeptides assume various combinations of type II beta-turns, gamma-turns, and gamma 1-turns, but never type I beta-turns. These investigations provide evidence that the tetrapeptide building blocks retain their preferred conformations in larger molecules and can be used to control the architecture of regions of such peptides.

Amino Acid Sequence↗
PubMed1998

Structure of segments of a G protein-coupled receptor: CD and NMR analysis of the Saccharomyces cerevisiae tridecapeptide pheromone receptor.

Peptides representing both loop and the sixth transmembrane regions of the alpha-factor receptor of Saccharomyces cerevisiae were synthesized by solid-phase procedures and purified to near homogeneity. CD, nmr, and modeling analysis indicated that in aqueous media the first extracellular loop peptide E1(107-125), the third intracellular loop peptide I3(231-243), and the carboxyl terminus peptide I4(350-372) were mostly disordered. In contrast, the second extracellular loop peptide E2(191-206) assumed a well-defined structure in aqueous medium and the sixth transmembrane domain peptide receptor M6(252-269, C252A) was highly helical in trifluoroethanol/water (4:1), exhibiting a kink at Pro258. A synthetic peptide containing a sequence similar to that of the sixth transmembrane domain of a constitutively active alpha-factor receptor M6(252-269, C252A, P258L) in which Leu replaces Pro258 exhibited significantly different biophysical properties than the wild-type sequence. In particular, this peptide had very low solubility and gave CD resembling that of a beta-sheet structure in hexafluoroacetone/water (1:1) whereas the wild-type peptide was partially helical under identical conditions. These results would be consistent with the hypothesis that the constitutive activity of the mutant receptor is linked to a conformational change in the sixth transmembrane domain. The study of the receptor segments also indicate that peptides corresponding to loops of the alpha-factor receptor do not appear to assume turn structures.

Amino Acid Sequence↗
PubMed1998

Eighteen years' experience with neonatal Hirschsprung's disease treated by endorectal pull-through without colostomy.

METHODS: In the past 18 years, the authors have treated 84 patients with Hirschsprung's disease. Of these, 43 patients were under 1 month of age and underwent endorectal pull-through without colostomy. Some have undergone follow-up for as long as 18 years. RESULTS: Thirty-four of these 43 (79%) newborn patients were available for follow-up. Twenty-two were totally continent. The remaining 12 have normal sphincter tone. Of the 41 patients above 1 month of age, 34 (83%) were available for follow-up. Some have undergone follow-up for as long as 18 years. CONCLUSION: Twenty-two of this latter group (79%) have normal bowel control.

Adolescent↗
PubMed1998

Schizosaccharomyces pombe isp4 encodes a transporter representing a novel family of oligopeptide transporters.

We have recently cloned an oligopeptide transport gene from Candida albicans denoted OPT1. This gene showed significant sequence similarity to three open reading frames (ORFs) with no previously established function: isp4 from Schizosaccharomyces pombe and Saccharomyces cerevisiae YJL212C and YPR194C, identified during the genome project. The S. pombe gene isp4 was originally identified by Sato et al. as a gene that was upregulated through nitrogen starvation induction of meiosis. However, an isp4delta strain exhibited a wild-type phenotype with respect to sexual differentiation. We have found that the same isp4delta strain is deficient in tetrapeptide transport activity as measured by its resistance to toxic tetrapeptides, by its inability to accumulate a radiolabelled tetrapeptide and by the inability to use tetrapeptides as a sole source of an amino acid to satisfy an auxotrophic requirement. Similarly, we found that the ORF YPR194C from S. cerevisiae encodes an oligopeptide transporter. Sequence analyses as well as physiological evidence has led us to propose that the proteins encoded by isp4 and the genes identified from S. cerevisiae and C. albicans comprise a new group of transporters specific for small oligopeptides, which we have named the OPT family.

Amino Acid Sequence↗
PubMed1998

PTR3, a novel gene mediating amino acid-inducible regulation of peptide transport in Saccharomyces cerevisiae.

We have isolated and characterized the Saccharomyces cerevisiae PTR3 gene by functional complementation of a mutant deficient for amino acid-inducible peptide transport. PTR3 is predicted to encode a protein of 678 amino acids that exhibits no similarity to any other protein in the database. Deletion of the PTR3 open reading frame pleiotropically reduced the sensitivity to toxic peptides and amino acid analogues. Initial rates of radiolabelled dipeptide uptake demonstrated that elimination of PTR3 resulted in the loss of amino acid-induced levels of peptide transport. PTR3 was required for amino acid-induced expression of PTR2, the gene encoding the dipeptide/tripeptide transport protein, but was not necessary for nitrogen catabolite repression of peptide import or PTR2 expression. It was determined that PTR3 also modulates expression of BAP2, the gene encoding the branched-amino acid permease. Furthermore, we present genetic evidence that suggests that PTR3 functions within a novel regulatory pathway that facilitates amino acid induction of the PTR system.

Amino Acid Transport Systems↗
PubMed1998

Structure-function analysis of the Saccharomyces cerevisiae tridecapeptide pheromone using alanine-scanned analogs.

Twenty-six peptide analogs of the Saccharomyces cerevisiae alpha-factor, a tridecapeptide mating pheromone (W1H2W3L4Q5L6K7p8G9Ql0P11M12Y13) with either L- or D-alanine replacement of each amino acid residue (Ala-scanned) and with the isosteric replacement of methionine at position 12 by norleucine, were synthesized, purified to homogeneity and assayed for biological activity and receptor binding. Two new and effective antagonists, [D-Ala3,Nle12]alpha-factor and [D-Ala4,Nle12]alpha-factor, were found among the series, and the [D-Ala10,Nle12]alpha-factor demonstrated a marked ability to increase the biological activity of [Nle12]alpha-factor without having any effect by itself. One analog, the [L-Ala1 alpha-factor, showed a 3-fold increase in bioactivity over the [Nle12]alpha-factor, although its binding to the alpha-factor receptor was about 70-fold less than [Nle12]alpha-factor. Residues near the carboxyl terminus contributed more strongly to receptor binding than other residues, whereas those near the amine terminus of the alpha-factor played an important role in signal transduction. The effect of insertion of D-Ala residues at positions 7, 8, 9 and 10 on bioactivity and receptor binding of the peptide suggested a specific positioning role of the central loop in establishing optimal contacts between the receptor and the ends of the pheromone. We conclude that the alpha-factor may be divided into segments with dominant roles in forming the biologically active pheromone conformation, in receptor binding and in initiating signal transduction. The discovery of such relationships was made possible by the systematic variation of each residue in the peptide and by the testing of each analog in highly defined biological and binding assays.

Alanine↗