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Biomedical subjects

G A Mueller

Publications and source records attributed to G A Mueller.

13 recordsLinked to original sources
PubMed1999

A robust and cost-effective method for the production of Val, Leu, Ile (delta 1) methyl-protonated 15N-, 13C-, 2H-labeled proteins.

A selective protonation strategy is described that uses [3-2H] 13C alpha-ketoisovalerate to introduce (1H-delta methyl)-leucine and (1H-gamma methyl)-valine into 15N-, 13C-, 2H-labeled proteins. A minimum level of 90% incorporation of label into both leucine and valine methyl groups is obtained by inclusion of approximately 100 mg/L alpha-ketoisovalerate in the bacterial growth medium. Addition of [3,3-2H2] alpha-ketobutyrate to the expression media (D2O solvent) results in the production of proteins with (1H-delta1 methyl)-isoleucine (> 90% incorporation). 1H-13C HSQC correlation spectroscopy establishes that CH2D and CHD2 isotopomers are not produced with this method. This approach offers enhanced labeling of Leu methyl groups over previous methods that utilize Val as the labeling agent and is more cost effective.

Carbon Isotopes↗
PubMed1998

Tertiary structure of the major house dust mite allergen Der p 2: sequential and structural homologies.

Sensitization to indoor allergens, especially those of the house dust mite, is strongly correlated with the development of asthma. We report the tertiary structure of the major house dust mite allergen, Der p 2, determined by NMR methods. The structure of Der p 2 is a beta-barrel and is composed of two three-stranded antiparallel beta-pleated sheets. This arrangement of beta-strands is similar to the immunoglobulin fold with respect to the orientation of the two sheets and the interactions of the strands. However, the three-dimensional structure of Der p 2 aligns equivalently with a number of proteins from different families within the immunoglobulin superfamily. The structural homology with the highest significance score from analysis by DALI is to Der f 2. Although Der p 2 and Der f 2 are 87% identical in amino acid sequence, they align in three dimensions rather poorly (4.85 A RMSD; Z-score, 8.58). This unexpected finding is likely due to the different solution conditions used during structure determination by NMR for both proteins. While the structural comparisons did not elucidate a clear homologue for the function of Der p 2 in mites, we report that Der p 2 is sequentially homologous to esr16. This is a protein from moths that is expressed coincident with molting. Thus, this homology has important ramifications for the study of mite allergy. The structure of Der p 2 provides a useful tool in the design of recombinant immunotherapeutics for the group 2 allergens.

Allergens↗
PubMed1998

Future directions for allergen immunotherapy.

Over the last 30 years several approaches to modify immunotherapy have been tested, including allergoids, alum precipitation, and most recently peptides. However, none of these have replaced the traditional regimens. Over the same period our scientific understanding of allergic disease has been transformed. Today it is possible to identify and monitor changes occurring during treatment and to target many different aspects of the immune system. Recombinant technology provides a powerful technique both for sequencing proteins and producing allergens in commercial quantities. The recombinant proteins can be modified by site-directed mutagenesis so as to decrease their reactivity with IgE antibodies while maintaining reactivity with T cells. Knowledge of the tertiary structure of allergens will make it simpler to identify and change surface epitopes. A completely different approach is to use plasmids to introduce the genes for an allergen. The strength of this technique is that the plasmid can be designed to control expression and also to influence the cytokine profile of the response or the isotype of antibodies produced. Finally, different adjuvants can be used with proteins to alter the response. These include IL-12, immunostimulatory sequences of DNA, and bacterial proteins such as those used in HibVax. It is now possible to identify the cells that control the immune response to allergens and to design treatments that will either downregulate or change the response of T cells. The challenge is to transform this information into an effective treatment for allergic disease.

Allergens↗
PubMed1998

Complementation of Der P 2-induced histamine release from human basophils sensitized with monoclonal IgE: not only by IgE, but also by IgG antibodies directed to a nonoverlapping epitope of Der p 2.

The interaction of free allergen with two (or more) IgE molecules bound to the high-affinity receptor for IgE (FcepsilonRI) on mast cells and basophilic granulocytes results in the release of inflammatory mediators. The role of allergen-specific IgG antibodies in the allergic reaction in human beings is less clear. We produced two chimeric IgE antibodies, hIgE-Dp2A and hIgE-Dp2B, directed to two nonoverlapping epitopes (A and B) of the house dust mite allergen Der p 2. Chimeric IgG1 and IgG4 variants of these antibodies were produced also. Basophil activation by the house dust mite allergen Der p 2 was induced after sensitization of basophils with a mixture of chimeric hIgE-Dp2A and hIgE-Dp2B antibodies but not after sensitization by the individual IgE antibodies alone. Basophil activation was also shown after sensitization with hIgE-Dp2A and stimulation with Der p 2 incubated with hIgG1-Dp2B or hIgG4-Dp2B antibodies. Both IgE and IgG antibodies directed to the other nonoverlapping epitope complemented the sensitization by the hIgE-Dp2A antibody. Nonsensitized basophils were not activated by the Der p 2/hIgG-Dp2 mixtures. These results indicate that allergen-specific IgG can complement an IgE-dependent reaction and therefore under certain conditions can act as an anaphylactic antibody.

Anaphylaxis↗
PubMed1997

Expression and secondary structure determination by NMR methods of the major house dust mite allergen Der p 2.

There exists a strong correlation between asthma and sensitization to indoor allergens. This study reports on the secondary structure of the major house dust mite allergen Der p 2, determined using heteronuclear NMR methods. The DNA was subcloned from the yeast expression vector pSAY1 into the high yield bacterial expression vector pET21a, resulting in yields of 50 mg/liter. The recombinant protein was shown to have immunoreactivity comparable with that of the natural mite protein using competitive inhibition enzyme-linked immunosorbent assay (ELISA) and a modified monoclonal radioallergosorbent test (RAST). The secondary structure was determined by examining chemical shifts, short and long range NOESYs, JHN-HA coupling constants, and amide exchange rates. From these data, it is clear that Der p 2 is composed of beta-sheets and random coil. Based on long range distance constraints, a number of beta-strands were aligned into two three-stranded, anti-parallel beta-sheets.

Allergens↗
PubMed1997

Mouse/human chimeric IgG1 and IgG4 antibodies directed to the house dust mite allergen Der p 2: use in quantification of allergen specific IgG.

BACKGROUND AND OBJECTIVE: Chimeric mouse/human monoclonal IgG1 and IgG4 antibodies were developed against the house dust mite allergen Der p 2. These chimeric IgG antibodies, hIgG1-Dp2 A and hIgG4-Dp2 A, have the same binding characteristics as the previously reported chimeric hIgE-Dp2 A and are composed of the heavy chain variable domains and light chains of the original murine monoclonal antibody 2B12, whereas the heavy chain constant domains have been replaced by the human IgG1 or IgG4 heavy chain. The expression level of hIgG1-Dp2 A and hIgG4-Dp2 A was 1 and 3.5 microg/mL, respectively. METHODS AND RESULTS: Since all IgG in these culture supernatants is allergen-specific, they are useful reference reagents and enable the calculation of the amount of allergen specific IgG1 and IgG4 antibodies in absolute IgG amounts. The results obtained with two panels of sera from patients in immunotherapeutic treatment were evaluated and compared in Der p 2 IgE, IgG1 and IgG4 RAST and with reversed IgG4 RAST using labelled purified Der p 2. Close agreement between the results for the two IgG4 assays was found. CONCLUSION: With these chimeric reference reagents the quantities of isotype specific antiallergen antibodies can be calculated and compared.

Animals↗
PubMed1994

The role of cytoskeletal proteins in neutrophil emigration during pneumonia in rabbits.

The cytoskeletal proteins, actin and tubulin, are critical in modulating many aspects of the structural, mechanical, and biochemical properties of cells. This study determined if rearrangements of microtubules or filamentous actin were necessary for neutrophil margination within the pulmonary microvasculature or emigration into the alveolar spaces in response to Streptococcus pneumoniae. Microtubule assembly was inhibited using colchicine, and F-actin depolymerization was inhabited using phalloidin. Anesthetized rabbits received an intrabronchial instillation of S. pneumoniae either after intravenous pretreatment with colchicine (1 mg/kg every 2 h) or combined with TRITC-phalloidin (2 microM in instillate). Four hours later, the lungs were fixed and removed. The results show that the intravenous injection of colchicine caused a rapid decrease in circulating neutrophil counts, most likely caused by sequestration within the pulmonary microvasculature, that gradually recovered. In the pneumonic region, colchicine inhibited neutrophil emigration by 74 +/- 5%, but it did not prevent the stimulus-induced increase in margination. Phalloidin inhibited neutrophil emigration by 83 +/- 4%. These studies suggested that microtubule reassembly occurs during neutrophil transit through the normal pulmonary microvasculature and that it is required for migration but not sequestration during pneumonia. Rearrangement of actin filaments in lung cells but not neutrophils are required for neutrophil emigration induced by S. pneumoniae.

Actins↗
PubMed1992

Pediatric pulmonary function testing in asthma.

Pulmonary function testing is an important tool in the management of asthma. Lung function can be readily assessed in both the office and patient's home. This article reviews spirometry, peak flow meters, and bronchial challenge testing. Their interpretation and clinical use are presented as well.

Albuterol↗
PubMed1971

The effects of varying concentrations of colchicine on the progression of grasshopper neuroblasts into metaphase.

The effects of four concentrations of colchicine (2.5 x 10(-7), x 10(-5), x 10(-3), and x 10(-2)M) on the cell cycle of grasshopper neuroblasts have been determined by direct observations on living cells. The lowest concentration, 2.5 x 10(-7)M, does not completely disorganize the spindle but does retard its action. The three higher concentrations disorganize the spindle, so that all cells reaching metaphase are blocked in a c-mitotic condition throughout the period of observations (308 min at 38 degrees C, the minimum duration of the cell cycle in untreated neuroblasts). Continuous treatment with all concentrations reduces the rate at which neuroblasts enter metaphase, the extent of the reduction being a function of increasing concentration and time of exposure. After a short exposure to 2.5 x 10(-5)M colchicine, the neuroblasts recover from the inhibiting effects on progression through the cycle to metaphase, but they show no recovery from the inhibiting effects on spindle formation for more than 3 hr. Apparent stimulation of progression rate occurs early in exposure to all concentrations and during recovery from a short exposure to 2.5 x 10(-5)M. Morphological alterations in the chromatin of telophase, interphase, and prophase cells are induced by the higher concentrations of colchicine. The data indicate that caution should be exercised in the use of colchicine for determining cell cycle duration and/or the effects of physical and chemical agents on the cycle.

Animals↗