Biomedical subjects

J M Becker

Publications and source records attributed to J M Becker.

At least 73 records · Page 4Linked to original sources

PubMed1995

Lipid-mediated a-factor interactions with artificial membranes.

Our understanding of the cellular export of a-factor and its interaction with the receptor do not yet allow for a description of the phenomena on a molecular level. Synthesis of a-factor analogs and biophysical studies of the lipopeptides in the presence of artificial membranes provide insights which can be analyzed with respect to the biological potency of the molecules. It is through the study of the interaction of the lipopeptides with membranes at varying levels of complexity that we will be able to develop a molecular description of the biological processes.

Alkyl and Aryl Transferases↗

PubMed1995

A recognition component of the ubiquitin system is required for peptide transport in Saccharomyces cerevisiae.

Peptide transport in Saccharomyces cerevisiae is controlled by three genes: PTR1, PTR2, and PTR3, PTR1 was cloned and sequenced and found to be identical to UBR1, a gene previously described as encoding the recognition component of the N-end-rule pathway of the ubiquitin-dependent proteolytic system. Independently derived ubr1 mutants, like ptr1 mutants, were unable to transport small peptides into cells. Concomitantly, ptr1 mutants, like ubr1 mutants, were unable to degrade an engineered substrate of the N-end-rule pathway. Further, ptr1 mutants did not express PTR2, a gene encoding the integral membrane component required for peptide transport in S. cerevisiae. These results establish a physiological role for a protein previously known to be required for the degradation of N-end-rule substrates. Our findings show that peptide transport and the ubiquitin pathway--two dynamic phenomena universal to eukaryotic cells--share a common component, namely UBR1/PTR1.

Base Sequence↗

PubMed1995

The PTR family: a new group of peptide transporters.

The transport of peptides into cells is a well-documented biological phenomenon which is accomplished by specific, energy-dependent transporters found in a number of organisms as diverse as bacteria and humans. Until recently, the majority of peptide transporters cloned and characterized were found to be proteins of the ATP-binding cassette (ABC) family. We report the identification of a new family of peptide transporters, which we call the PTR family. This group of proteins, distinct from the ABC-type peptide transporters, was uncovered by sequence analyses of a number of recently discovered peptide transport proteins. Alignment of these proteins demonstrated a high number of identical and similar residues and identified conserved glycosylation and phosphorylation sites, as well as a structural motif unique to this group of proteins. Cluster analysis among the proteins indicated these sequences were indeed related and could be further divided into two subfamilies. A phylogenetic analysis of these new peptide transport sequences, compared to over 50 other peptide and membrane-bound transporters, showed that these proteins comprise a distinct, separate group of proteins.

Amino Acid Sequence↗

PubMed1995

Synthesis of alpha-factor analogues containing photoactivatable and labeling groups.

Analogues of alpha-factor, Saccharomyces cerevisiae tridecapeptide mating pheromone (H-Trp-His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr-OH), containing both p-benzoyl phenylalanine (Bpa), a photoactivatable group, and 3-(mono- or di-iodo-4-hydroxyphenyl)propanoic acid (iodinated HPP) or biotin as a tag, were synthesized using solid-phase methodologies on a [phenylacetamido]-methyl (PAM) resin. Bpa was introduced into the peptides using Bpa-hydroxybenzotriazole active ester during peptide chain assembly. Biotinylated alpha-factor analogues were prepared by assembling the desired peptide on the resin, and then reacting a specific amino group either with the symmetrical anhydride of biotin or with biotin using BOP as the activating agent prior to anhydrous hydrogen fluoride cleavage. Iodinated HPP was incorporated by acylating free peptides with Bolton-Hunter reagent (3-[diiodo-4-hydroxyphenyl]propanoic acid hydroxysuccinimide ester) in N,N-dimethylformamide and borate buffer (pH 8.0) solutions. Purification of all peptides to 98% or greater homogeneity was accomplished by high-performance liquid chromatography on a reversed-phase mu-Bondapak C18 column with acetonitrile/water/trifluoroacetic acid as the mobile phase. All products were characterized by amino acid analysis and fast atom bombardment mass spectrometry. Two analogues, alpha-(diiodotyrosine)-His-Bpa-Leu-Gln-Leu-Arg-Pro-Gly-Gln-Pro-Nle-Tyr-O H, and epsilon-(diiodo-HPP)-Lys-His-Bpa-Leu-Gln-Leu-Arg-Pro-Gly-Gln-Pro-Nle-Tyr -OH, were one twentieth to one-fortieth as active as a alpha-factor, and exhibited approximately one order of magnitude lower affinity to the alpha-factor receptor. The results suggest that these two analogues are alpha-factor agonists and that they can be used as probes of the alpha-factor receptor.

Amino Acid Sequence↗

PubMed1995

Studies on conformational consequences of i to i + 3 side-chain cyclization in model cyclic tetrapeptides.

In an effort to explore the effect of ring size on the biologically active conformation of cyclic analogs of the mating pheromone alpha-factor (WHWLQLKPGQPMY) from Saccharomyces cerevisiae, eight cyclic tetrapeptides corresponding to the KPGQ portion of alpha-factor were synthesized. These N-alpha-acetyl/carboxyl amide terminal cyclic tetrapeptides were prepared on a 4-methylbenzhydrylamine resin using orthogonal Boc, Fmoc, OFm and OtBut protecting groups and HOBt-DIPC accelerated active esters or urethane-protected N-carboxyanhydrides. On-resin cyclization of the side-chain amino and carboxyl groups of the first and fourth residues, respectively, was performed with the BOP reagent to generate lactams containing 14-18 atoms. HF cleavage resulted in two products, the desired cyclic tetrapeptide and a major side product. All peptides were purified to near homogeneity (> 99%) by using reversed-phase HPLC and were characterized by FBMS and 1H NMR. Certain constrained cyclic tetrapeptides appear to be a mixture of isomers at room temperature as evidenced by HPLC and NMR. The major side product has been identified as a cyclo dimer, obtained as a consequence of interchain cyclization on the resin. CD analysis in several solvents gives evidence that some of the cyclic tetrapeptides exist in beta-turn conformations.

Amino Acid Sequence↗

PubMed1995

Reduced virulence of Candida albicans mutants affected in multidrug resistance.

Disruption of a multidrug resistance gene (CaMDR1) in Candida albicans resulted in mutant strains that colonized mouse kidneys to very high levels but were markedly reduced in their virulence. No obvious differences in several properties related to colonization and dissemination were noted among MDR+ or mdr- strains. These results suggest that specific fungal efflux pumps play a role in fungal pathogenicity.

ATP-Binding Cassette Transporters↗

PubMed1995

Fungal lipopeptide mating pheromones: a model system for the study of protein prenylation.

In a variety of fungal species, mating between haploid cells is initiated by the action of peptide pheromones. The identification and characterization of several fungal pheromones has revealed that they have common structural features classifying them as lipopeptides. In the course of biosynthesis, these pheromones undergo a series of posttranslational processing events prior to export. One common modification is the attachment of an isoprenoid group to the C terminus of the pheromone precursor. Genetic and biochemical investigations of this biosynthetic pathway have led to the elucidation of genes and enzymes which are responsible for isoprenylation of other polypeptides including the nuclear lamins, several vesicular transport proteins, and the oncogene product Ras. The alpha-factor of Saccharomyces cerevisiae serves as a model for studying the biosynthesis, export, and bioactivity of lipopeptide pheromones. In addition to being isoprenylated with a farnesyl group, the alpha-factor is secreted by a novel peptide export pathway utilizing a yeast homolog of the mammalian multidrug resistance P-glycoprotein. The identification of putative lipopeptide-encoding loci within other fungi, including the human immunodeficiency virus-associated opportunistic pathogen Cryptococcus neoformans and the plant pathogen Ustilago maydis, has stimulated much interest in understanding possible roles for pheromones in fungal proliferation and pathogenicity. Knowledge of variations within the processing, export, and receptor-mediated signal transduction pathways associated with different fungal lipopeptide pheromones will continue to provide insights into similar mechanisms which exist in higher eukaryotes.

Amino Acid Sequence↗

PubMed1994

Molecular determinants of bioactivity of the Saccharomyces cerevisiae lipopeptide mating pheromone.

The a-factor of Saccharomyces cerevisiae (YIIKGVF-WDPAC(Farnesyl)-OCH3) is a peptide pheromone in which post-translational modification with a farnesyl isoprenoid and carboxyl methyl group is required for export and bioactivity. Truncated and carboxyl-terminal modified analogs of the a-factor were synthesized in order to determine the effect of such modifications on bioactivity. Bioactivity studies on carboxyl-terminal analogs in which the chirality, the cysteine thioether, and the carboxyl ester were varied in an attempt to study the influence of topology on a-factor activity indicate that the hydrophobicity conferred by the farnesyl moiety and not its specific spatial orientation is a key determinant of a-factor potency. Analyses on truncated a-factors suggest that sequential removal of NH2-terminal residues leads to a gradient of potency loss, with some amino acids exhibiting a slightly greater contribution to bioactivity than others. Random oligonucleotide-targeted mutagenesis of the gene encoding a-factor was coupled to a biological screen to identify altered a-factor peptides which are secreted yet exhibit a loss of a-factor bioactivity. Transformants exhibiting this phenotype were examined to identify codon changes presumably responsible for the altered phenotype, thus indicating residues that may contribute significantly to a-factor bioactivity.

Amino Acid Sequence↗

PubMed1994

Nitric oxide producing neurons in the monkey and human digestive system.

Nitric oxide has been proposed as an inhibitory transmitter molecule that plays a role in muscle relaxation and vasodilation in the gastrointestinal tract. The present study analyzes the distribution of nitric-oxide-producing neurons in the monkey and human digestive system by means of nicotinamide-adenine-dinucleotide-phosphate-diaphorase histochemistry. This histochemical method is reliable and convenient for the visualization of neuronal nitric-oxide synthase, the enzyme responsible for nitric-oxide generation. In the gastrointestinal tract, nitric-oxide-synthase-related diaphorase activity was present in nerve fibers running throughout the muscular layer (circular > longitudinal) and in numerous ganglion cells and processes in the myenteric plexus of monkeys and humans. Labelled ganglion cells and fibers also were observed in the submucous plexus, although they were much less numerous than those seen in the myenteric plexus. In the submucosa, a few positive fibers were seen around blood vessels. In the mucosa, stained fibers were sparse at the base of the villi and crypts, whereas they were quite abundant in the muscularis mucosae, especially in the small intestine and colon. In the gallbladder (human), labelling was found in ganglion cells and processes of the innermost and outermost ganglionated plexuses. Stained fibers also were distributed to the muscular layer and, less abundantly, to the mucosa and vasculature. Labelled fibers were more abundant in the sphincter of Oddi (human) than in the gallbladder. In the monkey and human pancreas, nicotinamide-adenine-dinucleotide-diaphorase staining was seen mainly in ganglion cells and fibers of intrapancreatic ganglia, and in processes running among acini, around ducts and in the stroma. A moderate density of stained fibers also was distributed to the vasculature, whereas the islets showed few positive processes. Finally, double label experiments performed in the pancreas showed that the vast majority of neurons producing nitric oxide are immunoreactive for vasoactive intestinal peptide.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Consequences of altered isoprenylation targets on a-factor export and bioactivity.

Cysteine-containing amino acid sequences (CAAX, CC, and CXC; C is cysteine, A is any aliphatic amino acid, and X is any amino acid) are targets for the attachment of C15 (farnesyl) and C20 (geranylgeranyl) isoprenoids to peptides and proteins by specific prenyltransferases. Although much work has centered on the enzymatic mechanisms of these enzymes, the biological consequences of the differential isoprenylation they catalyze remain to be elucidated. Farnesylation of the a-factor mating pheromone of Saccharomyces cerevisiae is a known prerequisite for its biological activity and its secretion through a pathway utilizing the yeast STE6 protein, a homolog of the mammalian multidrug resistance (MDR) P-glycoprotein. We generated specific mutations in the a-factor gene to encode isoprenylation targets for geranylgeranylation [Cys-Val-Ile-Leu (CVIL) and Ser-Val-Cys-Cys (SVCC)] in place of the natural farnesylation motif [Cys-Val-Ile-Ala (CVIA)]. The a-factors containing these modified prenylation sites were successfully exported by a STE6-dependent mechanism. Furthermore, these peptides, as well as synthetic geranylgeranyl a-factor, retained bioactivity. Chromatographic comparisons of synthetic and biosynthetic pheromones suggest that, in vivo, a peptide substrate containing the geranylgeranylation target CVIL can be both farnesylated and geranylgeranylated. These results clearly demonstrate that in vivo (i) different prenyltransferases may recognize the same substrate; (ii) both farnesylated and geranylgeranylated a-factor peptides are substrates for export via STE6, a MDR-like protein; and (iii) farnesylated and geranylgeranylated pheromones are both biologically active.

ATP-Binding Cassette Transporters↗

PubMed1994

Does aggressive medical therapy for acute ulcerative colitis result in a higher incidence of staged colectomy?

BACKGROUND: Colectomy with ileal pouch-anal anastomosis is the operation of choice in patients with medically refractory ulcerative colitis. However, aggressive or prolonged medical treatment may result in the patient's needing an urgent operation in which a staged subtotal colectomy is necessary. OBJECTIVE: Our hypothesis is that the incidence of patients requiring a staged approach has increased, along with an increase in hospital stay and total hospital costs. DESIGN: We examined the medical records of 250 consecutive patients with ulcerative colitis who underwent ileal pouch-anal anastomosis between 1984 and 1993. RESULTS: Simultaneous colectomy and ileal pouch-anal anastomosis were performed in 196 patients (78%), while 54 patients (21.6%) required staged subtotal (78%) or partial colectomy (22%). Indications for initial colectomy included failure of medical therapy (42 patients [77.8%]), undifferentiated colitis (five patients [9.3%]), and perforation (six patients [11.1%]). An increase in the incidence of patients requiring staged colectomy during this period was observed (P < .05). Staged procedures led to a prolonged hospital course at a significantly greater total cost. CONCLUSION: We conclude that aggressive medical therapy of acute ulcerative colitis has increased the incidence of urgent staged colectomy with a resulting increase in morbidity, hospital stay, and cost and a less-optimal functional result.

Acute Disease↗

PubMed1994

Biophysical studies on fragments of the alpha-factor receptor protein.

The receptor for the alpha-factor mating pheromone of the yeast Saccharomyces cerevisiae consists of 431 amino acid residues and is a member of a family of membrane proteins predicted to have seven transmembrane helices. Fragments of the receptor corresponding to two of the transmembrane helices [residues 246-269 (M6) and 273-302 (M7)], two of the interhelical loops [residues 107-125 (E2) and 191-206 (E3)], and to a portion of the carboxyl terminus [residues 350-372 (CT)] were synthesized using solid-phase methodologies and purified to near homogeneity. CD was used to characterize the secondary structure of these peptides in trifluoroethanol (TFE), in TFE/water mixtures, in sodium dodecyl sulfate (SDS), and in the presence of dimyristoyl phosphatidylcholine (DMPC) liposomes. In TFE, M6 and M7 exhibited CD spectra consistent with highly helical peptides, whereas CT was partially helical. In contrast, E2 and E3 were either disordered or aggregated in this solvent. M6 did not partition well into DMPC vesicles whereas M7 remained helical. Both M6 and M7 assumed helical conformations in 25 mM SDS. The loop peptides and the carboxyl terminus peptide were either in a beta-structure or disordered in the presence of lipid. These findings represent the first biophysical evidence for conformations assumed by specific segments of the STE2 receptor protein.

Amino Acid Sequence↗

PubMed1994

NMR investigation of cyclo7,10[C7,X9,C10,Nle12] analogues of the alpha-factor from Saccharomyces cerevisiae.

The cyclo7,10[Cys7,Cys10,Nle12], cyclo7,10[Cys7,D-Ala9,Cys10,Nle12], and cyclo7,10[Cys7,L-Ala9,Cys10,Nle12] analogues of the alpha-factor mating pheromone (WHWLQLKPGQPMY) of the yeast Saccharomyces cerevisiae were studied in DMSO/water (80:20) and aqueous solution by nmr spectroscopy. In addition, the cyclo7,10[Cys7,D-Val9,Cys10,Nle12]alpha-fa ctor was examined in DMSO/water. Nuclear Overhauser effect (NOE) and NH d delta/dT data indicate that the cyclo7,10[Cys7,D-Val9,Cys10,Nle12]alpha-fa ctor adopts a type II beta-turn in DMSO/water and that the cyclo7,10[Cys7,D-Ala9,Cys10,Nle12]- and cyclo7,10[Cys7,L-Ala9,Cys10,Nle12]alpha-fa ctor analogues adopt type II and type I/III beta-turns, respectively, in both DMSO/water and aqueous solutions. In aqueous solution, residues 8 and 9 of the cyclo7,10[Cys7,Nle12] alpha-factor appear to adopt at least two distinct conformations, one of these being identified as a type I/III beta-turn. In contrast, the cyclo7,10[Cys7,Cys10,Nle12] alpha-factor appears to adopt predominately a type II beta-turn in DMSO/water. Quantitative NOE measurements of the cyclo7,10[Cys7,Cys10,Nle12]-, cyclo7,10[Cys7,D-Val9,Cys10,Nle12]-, and cyclo7,10[Cys7,L-Ala9,Cys10,Nle12] alpha-factors in DMSO/water were used to derive three-dimensional structures of the cyclo7,10[Cys7,Pro8,X9,Cys10] portion of these analogues.

Amino Acid Sequence↗

PubMed1994

Direct observation of cell wall glucans in whole cells of Saccharomyces cerevisiae by magic-angle spinning 13C-NMR.

Intact cells of Saccharomyces cerevisiae were examined as an aqueous paste by 13C-nmr spectroscopy with direct polarization and magic-angle spinning. The spectra obtained were highly resolved, showing numerous resonances in the 60-105 ppm range that were assigned to carbons of a liquid-like domain of the cell wall glucan. Assignments were confirmed by running the spectrum of S. cerevisiae in which the cell wall glucans were labeled with [13C] by feeding the cell [13C]galactose. The spectra indicate that the glucan in the cell wall of intact S. cerevisiae assumes a helical conformation and suggest that strain 17A fed with galactose preferentially incorporates the resulting glucose into beta (1-->3)-linkages.

Carbohydrate Sequence↗

PubMed1994

The occurrence of colon cancer in patients with known premalignant colonic mucosal diseases.

Both ulcerative colitis and familial polyposis are colonic mucosal diseases which are known to predispose to colon cancer. While colonoscopy is an accurate modality used in screening and surveillance for patients with these two diseases, patients continue to present with colon cancer with these known premalignant diseases. This study was conducted to ascertain why patients with known premalignant disease still develop life-threatening colon cancer and to assess the clinical profile and prognosis of patients with known ulcerative colitis (UC) and familial polyposis coli (FPC) who subsequently develop colon cancer. Total colectomy, mucosal proctectomy, and ileoanal pullthrough was performed on 367 patients with UC and FPC between January 1982 and March 1993 at our institution. Of these, 15 had invasive adenocarcinoma of the colon (4.1%) in addition to the primary disease. These 15 patients were studied in detail. The average duration of disease from diagnosis to definitive treatment of cancer was 17 years. Thirteen of the patients in this series had UC (87%), while only 2 had FPC (13%). Colonoscopy was used to make the diagnosis in 11 patients (73%), while the diagnosis was made only at the time of surgery in 3 (20%). Nine of the patients presented with a Dukes' B2 cancer or worse, representing 60% of the series. A high percentage had synchronous invasive cancers in this series--6 patients (40%). Despite the relatively high percentage of advanced cancers in this series, at a mean follow-up of 47.1 months, 14 of the 15 patients are still living. One patient has known recurrent disease while 1 has an elevated CEA with no other evidence of recurrence.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenocarcinoma↗

PubMed1994

In vivo toxicological effects of rel A antisense phosphorothioates in CD-1 mice.

To characterize the in vivo toxicity of phosphorothioate antisense oligonucleotides against rel A (p65 subunit of NF-kappa B transcription factor), forty-eight 6-week-old CD-1 mice were split into 4 groups (6/sex/group) receiving vehicle (phosphate-buffered saline) or doses of 50, 100, and 150 mg/kg of rel A antisense oligonucleotides intraperitoneally 3 times weekly for 2 weeks. Clinical signs of toxicity included weakness, and decreased motor activity and food consumption with body weight loss. Mortality occurred in 7 of 12 mice in the 150-mg/kg group and in 2 of 12 mice in the 100-mg/kg group, most of which died within the first 2 to 4 days of treatment. The remaining mice were necropsied on day 15. The major hematological finding was severe dose-dependent thrombocytopenia. The liver enzyme levels were mildly elevated in the serum of mid- and high-dose animals. At necropsy, increased spleen and liver weights were observed in treated mice, some of which also had mild pleural and/or peritoneal effusions. Histopathological examination revealed the likely cause of death to be acute renal failure due to renal cortical or tubular necrosis. Treatment-related changes were also found in the liver, spleen, bone marrow, and several other organs. In summary, the kidney, liver, and bone marrow (megakaryocytic lineage) were identified as the major target organs for toxicity with rel A antisense therapy.

Animals↗

PubMed1994

Does the histamine skin prick test identify patients at risk for toxicity after the ingestion of astemizole?

Astemizole is a widely prescribed nonsedating antihistamine that suppresses wheal and flare reactions from histamine prick testing. We report a two-year-old girl with a serum concentration-proven overdose of astemizole who nonetheless exhibited a significant wheal and flare reaction after histamine skin prick testing for at least 22 hours after the ingestion. These findings suggest that histamine skin prick testing should not be used as a screening test to evaluate whether an ingestion of astemizole has occurred.

Astemizole↗

PubMed1994

An Arabidopsis peptide transporter is a member of a new class of membrane transport proteins.

An Arabidopsis peptide transport gene was cloned from an Arabidopsis cDNA library by functionally complementing a yeast peptide transport mutant. The Arabidopsis plant peptide transporter (AtPTR2) allowed growth of yeast cells on dipeptides and tripeptides but not peptides four residues and higher. The plant peptide transporter also conferred sensitivity to a number of ethionine-containing, toxic peptides of chain length three or less and restored the ability to take up radiolabeled dileucine at levels similar to that of the wild type. Dileucine uptake was reduced by the addition of a variety of growth-promoting peptides. The sequence of a cDNA insert of 2.8 kb indicated an open reading frame encoding a 610-amino acid polypeptide (67.5 kD). Hydropathy analysis predicted a highly hydrophobic protein with a number of potential transmembrane segments. At the amino acid level, the Arabidopsis plant peptide transporter shows 24.6, 28.5, and 45.2% identity to the Arabidopsis nitrate-inducible nitrate transporter (CHL1), the rabbit small intestine oligopeptide transporter (PepT1), and the yeast peptide transporter (Ptr2p), respectively, but little identity to other proteins known to be involved in peptide transport. Root growth of Arabidopsis seedlings exposed to ethionine-containing toxic peptides was inhibited, and growth was restored by the addition of certain peptides shown to compete with dileucine uptake in yeast expressing the Arabidopsis transport gene. Consistent with the observed inhibition of root growth by toxic peptides, the peptide transporter is expressed in the roots of Arabidopsis seedlings. This study represents the characterization of a plant peptide transporter that is a member of a new class of related membrane transport proteins.

Amino Acid Sequence↗