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Biomedical subjects

P Whitley

Publications and source records attributed to P Whitley.

At least 19 recordsLinked to original sources
PubMed1998

Proline-induced disruption of a transmembrane alpha-helix in its natural environment.

alpha-Helix formation in globular proteins has been studied both theoretically and experimentally for decades, while a lack of both high-resolution structures and suitable experimental techniques has hampered the study of helices in membrane proteins. We have developed a new experimental approach, glycosylation mapping, where the active site of the lumenally exposed endoplasmic reticulum enzyme oligosaccharyl transferase is used as a point of reference against which the position of a transmembrane segment in the membrane can be measured. Here, we report an initial analysis of the helix-breaking properties of proline residues inserted in a transmembrane helix. We find that proline residues can break a transmembrane helix, but only when inserted near the end, and only when the helix is sufficiently long. The glycosylation mapping technique may be generally useful for determining the position of transmembrane helices in the membrane.

Amino Acid Sequence↗
PubMed1998

Cloning and characterisation of a group II allergen from the dust mite Tyrophagus putrescentiae.

The complete cDNA encoding a major allergen from the dust mite Tyrophagus putrescentiae, Tyr p 2, has been sequenced and expressed. A degenerate primer was designed to the N-terminal amino acid sequence of the 16-kDa protein. The complete cDNA sequence was achieved by using reverse transcriptase PCR, PCR+1, standard cloning and sequencing techniques. The cDNA of Tyr p 2 is 552 nucleotides in length from the start codon including 126 nucleotides after the stop codon up to the beginning of the poly(A) tail. The leader sequence consists of 15 amino acids. Regarding the predicted amino acid sequence, there are no potential N-glycosylation sites (N-X-S/T). The sequence showed similarity to group II allergens from other mite species, and some regions are completely conserved. To show that the cloned cDNA sequence was coding for an allergen, Tyr p 2 was expressed in Escherichia coli and shown to react with a T. putrescentiae-positive serum pool.

Allergens↗
PubMed1998

Contribution of disulphide bonds to antigenicity of Lep d 2, the major allergen of the dust mite Lepidoglyphus destructor.

To study the contribution of the 3 disulphide bonds in the major allergen Lep d 2 to the antigenic structure, site-directed mutagenesis was performed. Mutants with one or more cysteine residues altered were constructed with a histidine residue tag for purification purposes and expressed as recombinant proteins in E. coli. Seven mutants were analysed: 3 single mutants (Cys 8, Cys 21 and Cys 72), 3 double mutants (Cys 8-117, Cys 21-16 and Cys 72 77) and one mutant with all 6 cysteines altered (6 Cys). The evaluation of IgE reactivity in 10 allergic patients showed that the disulphide bond formed by cysteine 72 and 77 was the single most contributing bond to IgE binding. Mutants with disruption of the Cys 8-117 bond had a lesser reduction in IgE binding, even though this alteration seemed to influence the compact nature of Lep d 2. However, to abolish the IgE reactivity almost completely, all 6 cysteines had to be altered. A monoclonal antibody previously raised against Lepidoglyphus destructor showed a similar binding as human IgE with no reactivity to the Cys 72 77 or the 6 Cys mutant. Using skin prick test we found no reaction to the 6 Cys mutant at the concentrations tested (1-100 microg/ml) in an Lepidoglyphus destructor allergic patient, while the T-cell reactivity was preserved. The 6 Cys mutant of Lep d 2 may, after further evaluation, be a candidate molecule for improved immunotherapy of Lepidoglyphus destructor allergy.

Allergens↗
PubMed1998

Expression of two isoforms of Lep d 2, the major allergen of Lepidoglyphus destructor, in both prokaryotic and eukaryotic systems.

BACKGROUND: The dust mite Lepidoglyphus destructor is a major cause of allergic diseases among farmers. We have previously cloned and sequenced two isoforms of the major allergen Lep d 2 (formerly designated Lep d 1) and found significant homology to group 2 allergens of the house dust mite species Dermatophagoides. We now report on the production and characterization of recombinant Lep d 2. OBJECTIVE: We have expressed both isoforms in two different expression systems; a eukaryotic system, baculovirus in insect cells and a prokaryotic system, E. coli. We have compared the two systems in regard to production yields and immunoreactivity of the recombinant allergens. METHODS: The complete cDNA including the natural leader sequence was cloned into the pBlueBacIII transfer vector, and the rLep d 2 was produced as a secreted protein in baculovirus. For the expression in E. coli, the cDNA was cloned into the pET vector, and the rLep d 2 was produced with six C-terminal histidine residues. The purified recombinant allergens were tested for immunoreactivity with 10 sera from subjects allergic to Lepidoglyphus destructor and were compared with native Lep d 2 using inhibition immunoblotting. The ability of the recombinant allergens to release histamine from basophils was evaluated using a histamine release assay. RESULTS: Both expression systems produced immunoreactive recombinant allergens. They inhibited the binding of human sera to native Lep d 2 confirming their retained IgE binding properties. The yield of pure recombinant protein from the prokaryotic system was approximately 1 mg/L compared to the eukaryotic system which produced up to 4 mg/L in an adherent cell culture system. CONCLUSIONS: We have produced recombinant Lep d 2 in prokaryotic and eukaryotic expression systems which are comparable to the native allergen. Recombinant Lep d 2 might now be included in more extensive clinical studies to confirm its usefulness in the in vitro and the in vivo diagnosis of Lepidoglyphus destructor.

Allergens↗
PubMed1998

IgE binding capacity of synthetic and recombinant peptides of the major storage mite (Lepidoglyphus destructor) allergen, Lep d 2.

BACKGROUND: Lepidoglyphus destructor is an important non-pyroglyphid mite species in Europe and a dominant allergen in farming environments. The major allergen of L. destructor, Lep d 2, is a protein of 13.2 kD that is recognised by about 90% of sera RAST positive to this mite species. METHODS: The cDNA of two isoallergens of the Lep d 2 has previously been sequenced and the protein expressed in different protein expression systems. In order to map the B-cell epitopes, the full length protein and the truncated forms of the protein have been expressed in Escherichia coli as glutathione-S-transferase (GST) fusion proteins. Recombinant Lep d 2 fragments and synthetic overlapping 15 mer peptides spanning Lep d 2 were probed with sera from patients allergic to storage mite. RESULTS: The full-length (125 amino acids) GST fusion protein reacted strongly with patient IgE in Western blots and dot blots. Synthetic peptides failed to react with IgE antibodies from mite-allergic patients and the truncated fusion proteins displayed weak IgE-binding capacity. CONCLUSION: We conclude that there are no dominant linear IgE-binding epitopes in Lep d 2. Recombinant or synthetic Lep d 2 fragments may, however, be further evaluated as hypoallergenic candidate molecules for specific immunotherapy.

Allergens↗
PubMed1998

The design of arm pressure covers to alleviate pain in high G maneuvers.

HYPOTHESIS: Arm pain has been noted especially when increased coverage anti-G suits are worn in combination with PBG (pressure breathing during G) in high G centrifuge test runs. Arm pain has forced the termination of test runs and has the potential to cause a serious decrement in a pilot's ability to function effectively during high-G flight. Based on the theory that arm pain is caused by fluid pooling in the arm, four new pressure cover systems for the arm were designed and tested. METHODS: Seven test subjects wore each design in a series of runs in a dynamic flight simulator (DFS). Subjects experienced both gradual and rapid onset rate runs with G levels ranging from +3.0 G to +9.0 G. Data were collected on the maximum G's achieved, pain location, and pain intensity. Subjective responses on the comfort and function of each design and overall arm cover design preferences were also obtained via a questionnaire. RESULTS: Analysis of the DFS data, using a two-factor ANOVA (subjects, designs), revealed that each of the new designs provided significantly better protection from arm pain than the control condition (no protection) and that each of the designs significantly improved the maximum G level attained during rapid onset acceleration in the range of +1 G. CONCLUSION: No positive correlation was found between subject ranking of the four new design concepts at the conclusion of the study in terms of the pain reduction they provided and the pain levels reported during DFS testing.

Adult↗
PubMed1998

Evaluation of in vivo and in vitro quality of apheresis-collected RBC stored for 42 days.

BACKGROUND AND OBJECTIVES: New technological developments make it possible to collect red blood cells (RBCs) by apheresis, which allows for better product consistency and has the potential for improved RBC quality. The purpose of these studies was to evaluate the quality and consistency of units of RBCs collected by apheresis using the MCS+(R) machine (Haemonetics Corp., Braintree, Mass., USA). MATERIALS AND METHODS: Two studies were performed. In study 1 (n = 10), using containers and CP2D/AS-3 solutions from Medsep Corp. (Covina, Calif. USA), one-unit apheresis RBCs were compared to manually collected RBCs in a random crossover design. In study 2 (n = 12), 6 subjects had one unit collected, while the remaining 6 subjects had two units of RBCs collected with comparison to previously manually collected RBCs from the same donors. Haemonetics containers and solutions were used in study 2. RESULTS: Low RBC volume variability was found for the apheresis collections with a standard deviation of only 6 ml difference between actual and target volumes. Combining the data from the two studies (n = 21 pairs), at 42 days of storage, the apheresis units showed slightly lower hemolysis (0.44+/-0.26 vs. 0.61+/-0.50%), lower supernatant potassium levels (50+/-3 vs. 53+/-3 mEq/l), and improved tolerance to osmotic shock (47+/-3 vs. 49+/-3%) as compared to manual units (p < 0.05). There was no statistically significant difference in RBC ATP (3.0+/-0.6 vs. 2.9+/-0.5 micromol/g Hb) or in 24-hour percent recoveries (81+/-6 for apheresis vs. 81+/-4% for apheresis red cells). Apheresis RBC quality was not affected by the manufacturer (Haemonetics vs. Medsep) of solutions and containers. CONCLUSIONS: RBC units collected by apheresis demonstrated low variability in volume of RBC mass collected, and showed similar RBC properties as compared to manually collected RBCs after processing and after 42 days of storage.

Adolescent↗
PubMed1997

The complete cDNA sequence and expression of the first major allergenic protein of Malassezia furfur, Mal f 1.

For the first time the complete cDNA encoding a major allergen and novel protein of the yeast Malassezia furfur, Mal f 1, has been sequenced and expressed. The amino acid sequences of nine tryptic peptides of the protein were determined. Oligonucleotides were designed from these amino acid sequences. The cDNA sequence was obtained by hybridizing these primers to mRNA and enhancement by reverse-transcriptase PCR techniques. The cDNA is 1176 bp in length. It shows an open reading frame of 1050 bp coding for a protein of 38178 Da and a deduced amino acid sequence containing 350 residues. The hydropathy plot and the tryptic digest indicate that the first 22 amino acids represent a leader sequence determining a mature protein of 35 988 Da. The complete encoding cDNA was expressed as a maltose-binding protein fusion protein in Escherichia coli. The recombinant fusion protein reacted with our specific monoclonal antibody and with IgE from patients with atopic dermatitis.

Allergens↗
PubMed1997

Membrane-protein engineering.

Membrane proteins perform many of the essential functions required for life. They are often the targets of medicinal drugs and have many potential uses in biotechnological processes. Therefore our ability to understand them and manipulate their functions is both important and necessary to enable protein engineers to create 'designer' membrane proteins (that is, proteins designed to have desired properties).

Animals↗
PubMed1997

Evaluation of a new prestorage leukoreduction filter for red blood cell units.

BACKGROUND AND OBJECTIVES: Prestorage leukoreduction offers a variety of potential benefits and is becoming more commonly practiced. The LeukoNet prestorage leukoreduction filtration system is intended for leukoreduction of red blood cells and uses a vent to allow automatic drainage of red cells from the filter. MATERIALS AND METHODS: We studied the functional characteristics and the in-vivo and in-vitro properties of leukoreduced AS-1 Red Blood Cells prepared with this new system. Units of AS-1 Red Blood Cells were filtered at 4 degrees C through the LeukoNet filter 24-48 h after collection and stored under usual conditions for 42 days. Residual leukocytes were enumerated using a Nageotte chamber or with a polymerase chain reaction (PCR) technique. In the clinical trial (phase one), 21 donors had units stored with and without leukoreduction for 42 days; biochemical assays were done before and after storage, and 51Cr/99mTc red cell recovery studies at the end of the storage period. RESULTS: Leukocyte content after filtration was 3.2 +/- 2.6 x 10(4)/unit (n = 21), and all units had < 1 x 10(5) leukocytes (median: 3.8 x 10(4)). In-vivo paired studies showed no difference in 24-hour recovery (control: 82.1 +/- 5.8%; test: 82.9 +/- 6.0%). Hemolysis was halved with leuko-reduction (0.59 +/- 0.30 vs. 0.29 +/- 0.11%; p < 0.05), and glucose consumption was reduced by 5% compared to control units (p = < 0.05). Other biochemical parameters showed no differences. In the practical trial (phase two), filtration time was 41 +/- 23 min. With a residual leukocyte content of 6.6 +/- 4.9 x 10(4)/unit and 14 +/- 3% red cell loss (n = 84). Six additional units underwent leukocyte enumeration by PCR and had 2.6 +/- 1.1 x 10(4) residual leukocytes. CONCLUSIONS: Under the conditions studied, the LeukoNet leukoreduction filtration system produces about 4-5 log10 leukocyte content reduction.

Erythrocyte Transfusion↗
PubMed1997

Calmodulin point mutations affect Drosophila development and behavior.

Calmodulin (CAM) is recognized as a major intermediary in intracellular calcium signaling, but as yet little is known of its role in developmental and behavioral processes. We have generated and studied mutations to the endogenous Cam gene of Drosophila melanogaster that change single amino acids within the protein coding region. One of these mutations produces a striking pupal lethal phenotype involving failure of head eversion. Various mutant combinations produce specific patterns of ectopic wing vein formation or melanotic scabs on the cuticle. Anaphase chromosome bridging is also seen as a maternal effect during the early embryonic nuclear divisions. In addition, specific behavioral defects such as poor climbing and flightlessness are detected among these mutants. Comparisons with other Drosophila mutant phenotypes suggests potential CAM targets that may mediate these developmental and behavioral effects, and analysis of the CAM crystal structure suggests the structural consequences of the individual mutations.

Alleles↗
PubMed1996

A neurotransmitter transporter encoded by the Drosophila inebriated gene.

Behavioral and electrophysiological studies on mutants defective in the Drosophila inebriated (ine) gene demonstrated increased excitability of the motor neuron. In this paper, we describe the cloning and sequence analysis of ine. Mutations in ine were localized on cloned DNA by restriction mapping and restriction fragment length polymorphism (RFLP) mapping of ine mutants. DNA from the ine region was then used to isolate an ine cDNA. In situ hybridization of ine transcripts to developing embryos revealed expression of this gene in several cell types, including the posterior hindgut, Malpighian tubules, anal plate, garland cells, and a subset of cells in the central nervous system. The ine cDNA contains an open reading frame of 658 amino acids with a high degree of sequence similarity to members of the Na+/Cl(-)-dependent neurotransmitter transporter family. Members of this family catalyze the rapid reuptake of neurotransmitters released into the synapse and thereby play key roles in controlling neuronal function. We conclude that ine mutations cause increased excitability of the Drosophila motor neuron by causing the defective reuptake of the substrate neurotransmitter of the ine transporter and thus overstimulation of the motor neuron by this neurotransmitter. From this observation comes a unique opportunity to perform a genetic dissection of the regulation of excitability of the Drosophila motor neuron.

Amino Acid Sequence↗
PubMed1996

A 12-residue-long polyleucine tail is sufficient to anchor synaptobrevin to the endoplasmic reticulum membrane.

Synaptobrevin is a tail-anchored protein with a hydrophobic C-terminal transmembrane segment that inserts into the endoplasmic reticulum membrane independently of the SRP/Sec61p pathway. Here, we show that idealized hydrophobic segments composed of 11-17 leucines and 1 valine function as insertion signals in vitro, whereas shorter segments do not. These results suggest that there are no specific requirements beyond overall hydrophobicity for C-terminal endoplasmic reticulum insertion signals.

Amino Acid Sequence↗
PubMed1996

A nascent secretory protein may traverse the ribosome/endoplasmic reticulum translocase complex as an extended chain.

We have measured the minimum number of residues in a translocating polypeptide required to bridge the distance between the P-site in endoplasmic reticulum-bound ribosomes and the lumenally disposed active site of the oligosaccharyl transferase. The results suggest that a nascent chain may traverse the ribosome/translocase complex in a largely extended conformation, and that hydrophobic stop-transfer segments have a more compact, possibly alpha-helical conformation in the translocase.

Amino Acid Sequence↗
PubMed1996

Ala-insertion scanning mutagenesis of the glycophorin A transmembrane helix: a rapid way to map helix-helix interactions in integral membrane proteins.

Alanine insertions into the glycophorin A transmembrane helix are found to disrupt helix-helix dimerization in a way that is fully consistent with earlier saturation mutagenesis data, suggesting that Ala-insertion scanning can be used to rapidly map the approximate location of structurally and/or functionally important segments in transmembrane helices.

Alanine↗
PubMed1995

SecA-independent translocation of the periplasmic N-terminal tail of an Escherichia coli inner membrane protein. Position-specific effects on translocation of positively charged residues and construction of a protein with a C-terminal translocation signal.

We have shown previously that the 100-residue-long periplasmic N-terminal tail of the Escherichia coli inner membrane protein ProW can be translocated across the inner membrane in a sec-independent manner and that its translocation is blocked by the introduction of three positively charged residues near its C-terminal end (Whitley, P., Zander, T., Ehrmann, M., Haardt, M., Bremer, E., and von Heijne, G. (1994) EMBO J. 13, 4653-4661). We have now further analyzed the requirements for translocation of the N-terminal tail and found that the introduction of even a single arginine can block translocation. Position-specific differences in the effects on translocation of arginine insertions suggest that the C-terminal end of the N-terminal tail is more critical for translocation than the central and N-terminal regions. We also show that the N-terminal tail is translocated in a truncation mutant where a stop codon is placed immediately after the first transmembrane segment, provided that the transmembrane segment is flanked on its C-terminal end by positively charged residues. Thus, sec-independent translocation of a relatively large domain can be induced by a translocation signal located at the extreme C terminus of a protein.

ATP-Binding Cassette Transporters↗
PubMed1994

Synergistic insertion of two hydrophobic regions drives Sec-independent membrane protein assembly.

We have studied the membrane insertion of two proteins from the inner membrane of Escherichia coli, both with two transmembrane segments connected by a short periplasmic loop: the M13 procoat protein and a mutant "inverted" leader peptidase. Neither molecule depends on the Sec machinery for insertion. We show that the introduction of a charged residue in the second transmembrane segment completely blocks insertion of both proteins. In contrast, a Sec-dependent procoat mutant, where the periplasmic region has been lengthened, inserts into the membrane even in the presence of a charged residue in the second hydrophobic domain. In addition, a large deletion within the second transmembrane domain of the leader peptidase mutant allows membrane translocation, but only under conditions where the SecA protein is functional. Furthermore, we show that the first hydrophobic domain is required for insertion of the short periplasmic loop of the "inverted" leader peptidase. These results suggest that Sec-independent insertion occurs by a synergistic entry of the two neighboring hydrophobic domains into the lipid bilayer.

Bacterial Proteins↗