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J M Becker

Publications and source records attributed to J M Becker.

At least 55 records · Page 3Linked to original sources
PubMed1998

Altered expression of selectable marker URA3 in gene-disrupted Candida albicans strains complicates interpretation of virulence studies.

The ura-blaster technique for the disruption of Candida albicans genes has been employed in a number of studies to identify possible genes encoding virulence factors of this fungal pathogen. In this study, the URA3-encoded orotidine 5'-monophosphate (OMP) decarboxylase enzyme activities of C. albicans strains with ura-blaster-mediated genetic disruptions were measured. All strains harboring genetic lesions via the ura-blaster construct showed reduced OMP decarboxylase activities compared to that of the wild type when assayed. The activity levels in different gene disruptions varied, suggesting a positional effect on the level of gene expression. Because the URA3 gene of C. albicans has previously been identified as a virulence factor for this microorganism, our results suggest that decreased virulence observed in strains constructed with the ura-blaster cassette cannot accurately be attributed, in all cases, to the targeted genetic disruption. Although revised methods for validating a URA3-disrupted gene as a target for antifungal drug development could be devised, it is clearly desirable to replace URA3 with a different selectable marker that does not influence virulence.

Animals↗
PubMed1997

Novel modifications to the farnesyl moiety of the a-factor lipopeptide pheromone from Saccharomyces cerevisiae: a role for isoprene modifications in ligand presentation.

The a-factor of Saccharomyces cerevisiae is a dodecapeptide pheromone [YIIKGVFWDPAC(farnesyl)-OCH3] in which posttranslational modification with a farnesyl isoprenoid and carboxymethyl group is required for full biological activity. Utilizing novel synthetic techniques and a well-characterized array of biological assays, we prepared original modifications to the farnesyl moiety of the pheromone in order to assess the importance of this part of the lipopeptide for biological activity. Specifically, the 3-methyl group was replaced to create analogs containing the ethyl, vinyl, tert-butyl, and phenyl moieties at the 3-position of the farnesyl chain. Subsequent biological analyses demonstrated that all of these modifications render an active pheromone, with the vinyl and ethyl analogs exhibiting higher activity than the native a-factor. However, the level of activity varied with the modification; the bulkier and more hydrophobic groups (tert-butyl and phenyl) exhibited lower biological activity than the smaller moieties (ethyl and vinyl). Furthermore, two analogs with phenyl substitutions that differ only in the presumed isomerization of the allylic double bond show up to an 8-fold difference in bioactivity. It has previously been surmised that the role of isoprenoid additions is solely to target the attached polypeptides to membranes by increasing their hydrophobicity. However, these studies demonstrate that even modest structural changes to the isoprenoid can significantly affect biological activity. These results are clearly inconsistent with a simple hydrophobic role for the isoprenoid and instead illustrate that it plays an active role in mediating optimal a-factor/receptor interaction.

Butadienes↗
PubMed1997

Synthesis of prenylated peptides and peptide esters.

Modification of the cysteine sulfur in peptides and proteins to a thioether is a recently described posttranslational event that results in the incorporation of farnesyl and geranylgeranyl moieties. The increased lipophilicity accompanying these modifications often causes localization of the resulting protein to the membrane and may be essential for biological activity. Methods are described to chemically and biochemically synthesize farnesylated and geranylgeranylated peptides and proteins from microgram to gram quantities. Conditions for thioalkylation include acidic, neutral, and basic media. The ability to readily form peptidylthioethers will greatly facilitate studies of biologically important proteins and peptides containing isoprenyl moieties.

Esters↗
PubMed1997

Extent of smooth muscle resection during mucosectomy and ileal pouch-anal anastomosis affects anorectal physiology and functional outcome.

PURPOSE: In patients undergoing colectomy with ileal pouch-anal anastomosis, controversy exists regarding the necessity for and appropriate extent of rectal mucosal resection. Our aim was to assess histologically the extent of anorectal smooth muscle resected at the time of mucosal proctectomy and to correlate this with postoperative bowel and anal sphincter function. METHODS: Surgical specimens of 79 patients undergoing colectomy, mucosal proctectomy, and ileal pouch-anal anastomosis were examined histologically in a blinded fashion, and the content of smooth muscle in the mucosal proctectomy specimens was scored. Degree of smooth muscle resection was correlated with postoperative anorectal manometry and with functional outcomes, including stool frequency and nocturnal leakage of stool after 3 and 12 months of follow-up. RESULTS: Degree of smooth muscle loss correlated with decreased resting pressure of the internal anal sphincter as early as three months after surgery (r = -0.26; P = 0.03), and the correlation was even stronger after 12 months (r = -0.37; P = 0.005). Decreases in resting pressure were related, in turn, to increased stool frequency at 12 months (r = 0.32; P = 0.02), but stool frequency was also inversely related to volume of the ileal pouch (r = -0.27; P = 0.05). Multivariate analysis confirmed that resting pressure and pouch volume were both significant determinants of stool frequency. The likelihood of nocturnal stool leakage at 12 months was primarily a function of stool frequency (P < 0.01) but also increased with patient age (P < 0.02). CONCLUSIONS: These findings indicate that loss of resting pressure of the internal anal sphincter can be correlated with the extent of smooth muscle resection during rectal mucosectomy and that these factors, in turn, correlate with increased stool frequency and a greater likelihood of nocturnal stool leakage. Consequently, an optimum functional result requires care in identifying and preserving maximum anorectal smooth muscle during mucosectomy.

Age Factors↗
PubMed1997

Antisense expression of the peptide transport gene AtPTR2-B delays flowering and arrests seed development in transgenic Arabidopsis plants.

Previously, we identified a peptide transport gene, AtPTR2-B, from Arabidopsis thaliana that was constitutively expressed in all plant organs, suggesting an important physiological role in plant growth and development. To evaluate the function of this transporter, transgenic Arabidopsis plants were constructed expressing antisense or sense AtPTR2-B. Genomic Southern analysis indicated that four independent antisense and three independent sense AtPTR2-B transgenic lines were obtained, which was confirmed by analysis of the segregation of the kanamycin resistance gene carried on the T-DNA. RNA blot data showed that the endogenous AtPTR2-B mRNA levels were significantly reduced in transgenic leaves and flowers, but not in transgenic roots. Consistent with this reduction in endogenous AtPTR2-B mRNA levels, all four antisense lines and one sense line exhibited significant phenotypic changes, including late flowering and arrested seed development. These phenotypic changes could be explained by a defect in nitrogen nutrition due to the reduced peptide transport activity conferred by AtPTR2-B. These results suggest that AtPTR2-B may play a general role in plant nutrition. The AtPTR2-B gene was mapped to chromosome 2, which is closely linked to the restriction fragment length polymorphism marker m246.

Agrobacterium tumefaciens↗
PubMed1997

Position one analogs of the Saccharomyces cerevisiae tridecapeptide pheromone.

Analogs of the Saccharomyces cerevisiae alpha-mating factor [WHWLQLKPGQPMY], in which a variety of residues replaced Trp1 were synthesized and assayed for biological activity and receptor affinity. Analogs containing Gly or Leu or many different aromatic residues in position 1 of the peptide exhibited bioactivity in a growth arrest assay slightly greater than, or equal to, that of the parent pheromone, whereas the Glu1 and Lys1 analogs exhibited significantly lower bioactivity. Analogs with an aromatic replacement at position 1 had 3- to 6-fold lower receptor affinity than the parent peptide, whereas analogs with a hydrophilic residue at the N-terminus exhibited large reductions in receptor affinity with the peptide with Glu in position 1 showing a 120-fold reduction. N alpha-Acetylation had little effect on bioactivity but lowered receptor affinity by 20- to 40-fold. Amidation of the carboxyl terminus resulted in a 10-fold decrease in activity and a 160-fold decrease in receptor affinity. These results indicate that the alpha-factor receptor has a large hydrophobic binding pocket, possibly containing a negatively charged side-chain, which interacts with the N-terminus of alpha-factor. The lack of correlation between activity and binding and several analogs suggests that small residues near the N-terminus of alpha-factor may be very efficient in triggering isomerization of the receptor to its activated state in the first step of the signal transduction pathway.

Gene Expression↗
PubMed1996

Electrophoretic behavior of L- and D-alanine-scanning analogs of a yeast tridecapeptide pheromone in a fused-silica capillary.

Electrophoretic behavior of synthetic tridecapeptide diastereomers has been systematically investigated using a series of L-Ala- and D-Ala-scanning analogs of [Nle12] alpha-factor [WHWLQLKPGQP(Nle)Y], a tridecapeptide mating pheromone of Saccharomyces cerevisiae. The effects of buffer pH, buffer concentration, voltage, and temperature on diastereomer separation were tested. Among 13 pairs of diastereomers, those with L-Ala/D-Ala replacement in the middle of the peptide chain exhibited much higher diastereomeric resolution than those with identical replacement near the peptide termini. The fact that D-Ala9 and D-Ala12 homologs exhibited abnormal mobility differences compared to their L-diastereomers is probably related to the conformational restriction imposed by a Pro-D-Ala sequence. The results on the alpha-factor analogs represent the first observations of the influence of peptide secondary structure on mobility during capillary electrophoresis.

Amino Acid Sequence↗
PubMed1996

Preparation of radiolabeled peptides via an iodine exchange reaction.

Peptides labeled with radioactive 125I can be detected at the extremely low concentrations necessary for receptor binding studies and medical applications. Traditional methods of iodination often lead to inactive peptides due to excessive iodination, nonspecific iodination, or oxidative damage to the peptide. In this work these disadvantages are circumvented by labeling 127I containing peptides of a predetermined biological activity using a radioactive 125I exchange reaction with Na125I. Specific radioactivity up to 9.8 Ci/mmol was reached in a simple and efficient procedure.

Chromatography, High Pressure Liquid↗
PubMed1996

A superactive peptidomimetic analog of a farnesylated dodecapeptide yeast pheromone.

The S. cerevisiae a-factor, YIIKGVFWDPAC(s-farnesyl)-OCH3, is one of two peptide mating pheromones which mediate cell-cell communication in S. cerevisiae. We previously reported that replacing Gly5 with D-Ala led to a 4-6 fold increase in activity while the L-Ala5 homolog was 4 to 16-fold less active than the wildtype. To clarify the structural implications of these findings, we conformationally restricted the center of the pheromone by inserting gamma-lactam constraints in place of either the Lys4Gly5 or the Gly5Val6 dipeptide unit. Incorporation of (R)-3-amino-2-oxo-1-pyrrolidineacetic acid in place of Lys4Gly5 led to a super-active agonist which exhibited a 32-fold higher bioactivity than that of the a-factor. In contrast, an analog with (S)-3-amino-2-oxo-1-pyrrolidineacetic acid in place of Gly5Val6 is about 30 to 60-fold less active than the a-factor. These data strongly suggest that the a-factor adopts a reverse turn as its bioactive conformation.

Amino Acid Sequence↗
PubMed1996

Colonic ulceration associated with nonsteroidal anti-inflammatory drugs. Report of three cases.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are associated with a variety of gastrointestinal side effects. Effects on the large intestine have been reported with increasing frequency. Recognition of NSAID-induced colonic lesions has been confounded by variable clinical presentations, variable pathologic findings, and unfamiliarity of this entity among clinicians. We have recently seen three cases of NSAID-induced cecal ulcerations in patients undergoing right colectomy. A correct preoperative diagnosis was not made in our patients, one of whom presented with an acute abdomen and two in whom there was an inability to rule out carcinoma. The gross, radiographic, and histologic findings in each case consisted of a characteristic transverse ulceration with thin diaphragm-like scarring. NSAID-induced cecal ulcers can have a variety of presentations to the general surgeon, are likely to be misdiagnosed preoperatively, but may be recognized based on characteristic gross features evident by radiography and colonoscopy, along with a careful history. Review of recent literature suggests that laparotomy can be avoided when diagnosis is considered, but operation is indicated for complications, such as hemorrhage, obstruction, or perforation, and when carcinoma cannot be adequately excluded.

Abdomen, Acute↗
PubMed1996

Cloning of a second Arabidopsis peptide transport gene.

Previously, we reported the isolation of a peptide transport gene designated AtPTR2 from Arabidopsis thaliana by functional complementation of a yeast peptide transport mutant. We now report the isolation of a second peptide transport gene (AtPTR2-B) from Arabidopsis using the same approach. Similar to the effects of transferring AtPTR2-A (previously called AtPTR2), transfer of AtPTR2-B to yeast peptide transport mutants restored the ability to grow on di- and tripeptides but not peptides four residues or longer. However, unlike yeast mutants complemented with either the yeast PTR2 gene or the AtPTR2-A gene, transformants expressing AtPTR2-B were only partially sensitive to toxic peptides. Northern analysis showed that AtPTR2-B was constitutively expressed in all plant organs. Studies of the kinetics indicated that AtPTR2-A and AtPTR2-B have Km values of 47 and 14 microM, respectively, with Vmax values of 0.061 and 0.013 nmol mg-1 cell dry weight s-1, respectively, when dileucine was used as a substrate. AtPTR2-B is encoded on a 2.0-kb cDNA corresponding to a 585-amino acid protein (64.4 kD). Hydropathy analysis indicates that the protein is highly hydrophobic and suggests that there are 12 putative transmembrane segments. AtPTR2-B, like AtPTR2-A, shares significant similarity to a number of other proteins involved in transport of peptides into cells.

Amino Acid Sequence↗
PubMed1996

Probing the functional conformation of the tridecapeptide mating pheromone of Saccharomyces cerevisiae through study of disulfide-constrained analogs.

Analogs of the Saccharomyces cerevisiae alpha-mating factor, Trp-His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr, where Lys7 and Gln10 were replaced with Cys, Cys(CH3), or Ser, were synthesized using solid-phase procedures on a phenylacetamidomethyl resin. Cyclo7,10[Cys7,X9,Cys10,Nle12]alpha-factor , where X=D-Val, D-Ala, L-Ala and Gly, were prepared by on-resin cyclization using thallic trifluoroacetate in yields of 20-30%. Linear sulfhydryl-containing peptides were generated from their corresponding cyclic peptide by treatment with dithioerythritol in basic solution. In the linear analogs, replacement of both Lys7 and Gln10 with a cysteine residue resulted in an over 100-fold loss of the biological activity when compared with the native pheromone. The corresponding cyclic disulfides were 5-10-fold more active than their sulfhydryl-containing homologs, and cyclo7,10[Cys7,L-Ala9,Cys10,Nle12] alpha-factor was 50-fold more potent than linear analogs containing Ser or Cys(CH3) in positions 7 and 10. Binding competition studies indicated that all analogs had low affinity for the alpha-factor receptor and there was a poor correlation between binding and activity in a growth arrest assay. A cyclic analog in which residues 8 and 9 were replaced by 5-aminopentanoic acid was not biologically active. Based on NMR studies, all cyclic peptides have a higher tendency to form beta-turns spanning residues 7-10 than their less active linear counterparts. The results provide strong evidence that this beta-turn is important for optimal signal transduction by alpha-factor.

Amino Acid Sequence↗
PubMed1996

Prevention of postoperative abdominal adhesions by a sodium hyaluronate-based bioresorbable membrane: a prospective, randomized, double-blind multicenter study.

BACKGROUND: Postoperative abdominal adhesions are associated with numerous complications, including small bowel obstruction, difficult and dangerous reoperations, and infertility. A sodium hyaluronate and carboxymethylcellulose bioresorbable membrane (HA membrane) was developed to reduce formation of postoperative adhesions. The objectives of our prospective study were to assess the incidence of adhesions that recurred after a standardized major abdominal operation using direct laparoscopic peritoneal imaging and to determine the safety and effectiveness of HA membrane in preventing postoperative adhesions. STUDY DESIGN: Eleven centers enrolled 183 patients with ulcerative colitis or familial polyposis who were scheduled for colectomy and ileal pouch-anal anastomosis with diverting-loop ileostomy. Before abdominal closure, patients were randomly assigned to receive or not receive HA membrane placed under the midline incision. At ileostomy closure eight to 12 weeks later, laparoscopy was used to evaluate the incidence, extent, and severity of adhesion formation to the midline incision. RESULTS: Data were analyzed for 175 assessable patients. While only five (6 percent) of 90 control patients had no adhesions, 43 (51 percent) of 85 patients receiving HA membrane were free of adhesions (p < 0.00000000001). The mean percent of the incision length involved was 63 percent in the control group, significantly greater than the 23 percent observed in patients who received HA membrane (p < 0.001). Dense adhesions were observed in 52 (58 percent) of the 90 control patients, but in only 13 (15 percent) of the 85 receiving HA membrane (P < 0.0001). Comparison of the incidence of specific adverse events between the groups did not identify a difference (P > 0.05). CONCLUSIONS: This study represents the first controlled, prospective evaluation of postoperative abdominal adhesion formation and prevention after general abdominal surgery using standardized, direct peritoneal visualization. In this study, HA membrane was safe and significantly reduced the incidence, extent, and severity of postoperative abdominal adhesions.

Adenomatous Polyposis Coli↗
PubMed1995

Attenuated virulence of chitin-deficient mutants of Candida albicans.

We have analyzed the role of chitin, a cell-wall polysaccharide, in the virulence of Candida albicans. Mutants with a 5-fold reduction in chitin were obtained in two ways: (i) by selecting mutants resistant to Calcofluor, a fluorescent dye that binds to chitin and inhibits growth, and (ii) by disrupting CHS3, the C. albicans homolog of CSD2/CAL1/DIT101/KT12, a Saccharomyces cerevisiae gene required for synthesis of approximately 90% of the cell-wall chitin. Chitin-deficient mutants have no obvious alterations in growth rate, sugar assimilation, chlamydospore formation, or germ-tube formation in various media. When growing vegetatively in liquid media, the mutants tend to clump and display minor changes in morphology. Staining of cells with the fluorescent dye Calcofluor indicates that CHS3 is required for synthesis of the chitin rings found on the surface of yeast cells but not formation of septa in either yeast cells or germ tubes. Despite their relatively normal growth, the mutants are significantly less virulent than the parental strain in both immunocompetent and immunosuppressed mice; at 13 days after infection, survival was 95% in immunocompetent mice that received chs3/chs3 cells and 10% in immunocompetent mice that received an equal dose of chs3/CHS3 cells. Chitin-deficient strains can colonize the organs of infected mice, suggesting that the reduced virulence of the mutants is not due to accelerated clearing.

Animals↗
PubMed1995

Systematic analysis of the Saccharomyces cerevisiae alpha-factor containing lactam constraints of different ring size.

Eight cyclic analogs and corresponding linear homologs of the alpha-factor mating pheromone (WHWLQLKPGQPMY) of Saccharomyces cerevisiae were synthesized using solid-phase procedures on a phenylacetamidomethyl support. On-resin lactamization of the side chains of residues 7 and 10 to form rings containing from 14 to 18 atoms was effected by the BOP reagent. All peptides were highly homogeneous and gave expected molecular ions by FAB mass spectrometry. The constrained analogs had biological activities varying from 10% to less than 0.1% of that of [Nle12]-alpha-factor. In all cases, cyclic analogs with Glu in position 10 were more active than the homolog with Asp at this position. This trend was also found with the corresponding linear pheromones, suggesting that a gamma-carbonyl in position 10 is an important determinant of pheromone potency. The cyclic peptides had from 50- to 20000-fold lower affinities for the alpha-factor receptor than for [Nle12]-alpha-factor, as judged using a competition binding assay. Circular dichroism studies indicate that the cyclic lactam-containing region of cyclo7.10[Orn7, Glu10,Nle12]-alpha-factor retains a beta-turn-like structure similar to that found in the corresponding model tetrapeptide. The results show that covalently constrained analogs of the linear pheromone can maintain biological activity, despite binding poorly to the receptor, and indicate that a beta-turn-like structure in the center of the pheromone allows signal transduction.

Binding, Competitive↗
PubMed1995

Peptide immunoreactivities in the ganglionated plexuses and nerve fibers innervating the human gallbladder.

The mammalian gallbladder is innervated by a well-developed intrinsic neural network. However, little is known about the neurochemistry and organization of the innervation of this organ in humans. The aim of this study was to analyze the distribution of immunoreactivity (IR) for the neuropeptides, vasoactive intestinal polypeptide (VIP), neuropeptide Y (NPY), tachykinins (TK) and calcitonin gene-related peptide (CGRP) in the human gallbladder by means of immunohistochemistry. Neuropeptide-IRs are found in neurons and processes of the two ganglionated plexuses, i.e., the innermost plexus located in the lamina propria at the base of the mucosal folds, and the outermost plexus situated within the fibro-muscular layer. In these two plexuses, VIP-, NPY- and TK-IRs are present in ganglion cells and varicose fibers, whereas CGRP-IR is confined to nerve processes. VIP-IR is present in most, if not all, neurons. NPY- and TK-IRs are also found in many neurons. The densities of the peptide-IR nerves in the mucosa are NPY and VIP > TK >> CGRP, and in the fibro-muscular layer are NPY > VIP and TK > CGRP. The vasculature is richly innervated by NPY-IR nerves, which are mostly perivascular. CGRP-, VIP- and TK-IR processes are found only occasionally around blood vessels and in a paravascular position. Double-label studies demonstrated that a large number of VIP-containing neurons expresses NPY- or TK-IR. On the other hand, all neurons positive for either NPY- or TK-IR are immunostained for VIP. In agreement with these findings, most of the NPY-IR fibers in the lamina propria and fibro-muscular layer contain VIP-IR, and numerous TK-IR fibers are positive for VIP. However, the perivascular NPY-IR processes do not contain VIP-IR, suggesting an extrinsic origin. In addition, a population of TK-IR processes contains CGRP-IR and presumably originates from extrinsic sources, since CGRP/TK-IR intrinsic neurons could not be detected in the gallbladder. Peptide-IRs have a similar distribution in the neck, body and fundus of the gallbladder. No peptide-containing endocrine/paracrine cells are observed in the epithelium. The presence of peptide-IRs in the ganglionated plexuses and the abundance of peptidergic innervation suggest that peptides exert their effects on gallbladder function by acting directly on tissue targets and influencing intrinsic ganglion cells. Furthermore, the co-localization of more than one peptide in the same neuron raises the possibility that peptides are co-released upon stimulation and might interact at the same target.

Gallbladder↗