Validation of an ELISA method for quantification of the major Timothy grass pollen allergen Phl p 5a (BSP090).

Progress towards standardisation of allergen products has been made in recent years. Nevertheless, no standardised test method to quantify the allergen content of grass pollen allergen products is available at present. One aim of the BSP090 project was to validate a quantitative assay for a major Timothy grass (Phleum pratense) pollen allergen, Phl p 5. Qualification of a candidate ELISA system was performed with regard to range, robustness and cross-reactivity in preliminary studies. The assay specifically detected Phl p 5 with a quantification range from 3.9 ng/mL to 62.5 ng/mL. Suitability to quantify recombinant and natural Phl p 5 was further assessed in a collaborative study including 14 laboratories in Europe and the USA. Precision and accuracy of the assay was satisfactory with 93% of calculated Phl p 5 concentrations and 100% of total recoveries being within the ± 30% acceptance range. Similar results were obtained for spike recoveries, with exclusion of the lowest concentration spike, showing spike recoveries exceeding the acceptance range for six laboratories. Inter-assay (repeatability) and inter-laboratory (reproducibility) variability were satisfactory, in the format used in the present study. Robustness towards different statistical methods for data analysis was demonstrated. In conclusion, the assay can easily be established in routine testing and results of the preliminary testing and collaborative study support the proposal of the assessed Phl p 5-specific ELISA as a European Pharmacopoeia general method.

Author Correction: The influence of latitude, geographic distance, and habitat discontinuities on genetic variation in a high latitude montane species.

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The influence of latitude, geographic distance, and habitat discontinuities on genetic variation in a high latitude montane species.

Examining the factors that influence contemporary genetic patterns is important given the alarming rate at which natural environments are changing. In particular habitat fragmentation and climate change are expected to influence the distribution and diversity of natural populations. In this study we used both mitochondrial control region (mtDNA) and microsatellite data to answer the following questions about genetic diversity and divergence in mountain chickadees (Poecile gambeli) a resident bird species in western North America: (1) Do populations exhibit similar levels of genetic diversity across the range? (2) What is the genetic affinity of western populations in Oregon and Washington? (3) Do genetic patterns exhibit isolation by distance, or are genetic patterns more heavily influenced by habitat discontinuity? We tested the effects of isolation by distance and habitat distribution on genetic structure by analyzing 266 samples from 17 sites across western Canada and the United States. We found a near significant relationship between genetic diversity and latitude, however, our results indicate that overall, latitude is not a strong predictor of genetic diversity. Our analyses of populations in Oregon and Washington revealed a mismatch between patterns detected with mtDNA and microsatellite data. In particular, Washington clustered with the Coast Range/Cascades/Rocky Mountain mtDNA group, but with populations in southern Oregon/California based on microsatellite data. These results suggest the presence of a contact zone in Washington between the two mtDNA clades Coast Range/Cascades/Rocky Mountain and southern Oregon/California clades. Finally, our study revealed a greater effect of isolation by distance than isolation by habitat for both mtDNA and microsatellite data. Overall the isolation by distance signal was greater for mtDNA than microsatellite patterns. The greater signal of isolation by distance on mtDNA patterns likely reflects the strong effects of Pleistocene glaciations in shaping genetic patterns in western North America.

Sucl+ encodes a predicted 13-kilodalton protein that is essential for cell viability and is directly involved in the division cycle of Schizosaccharomyces pombe.

Sucl+ was originally identified as a DNA sequence that, at high copy number, rescued Schizosaccharomyces pombe strains carrying certain temperature-sensitive alleles of the cdc2 cell cycle control gene. We determined the nucleotide sequence of a 1,083-base-pair Sucl+ DNA fragment and S1 mapped its 866-nucleotide RNA transcript. The protein-coding sequence of the gene is interrupted by two intervening sequences of 115 and 51 base pairs. The predicted translational product of the gene is a protein of 13 kilodaltons. A chromosomal gene disruption of Sucl+ was constructed in a diploid S. pombe strain. Germinating spores carrying a null allele of the gene were capable of very limited cell division, following which many cells became highly elongated. The Sucl+ gene was also strongly overexpressed under the control of a heterologous S. pombe promoter. Overexpression of Sucl+ is not lethal but causes a division delay such that cells are approximately twice the normal length at division. These data suggest that Sucl+ encodes a protein which plays a direct role in the cell division cycle of S. pombe.

Cockroach allergens: function, structure and allergenicity.

Cockroach allergy is a widespread health problem in the world, associated with the development of asthma. The German and American cockroach species are important producers of a wide variety of allergens. Knowledge of their structure and function contributes to understand their role in allergy and to design tools for diagnosis and immunotherapy.

Gonadotrophin-releasing hormone and magnetic-resonance-guided ultrasound surgery for uterine leiomyomata.

OBJECTIVE: Magnetic-resonance-guided focused ultrasound is a novel, noninvasive technique of thermoablation for uterine leiomyomata. The hypothesis of this study was that pretreatment of leiomyomata with gonadotrophin-releasing hormone (GnRH) agonists would allow effective treatment of larger uterine leiomyomata, increasing the number of women who could benefit from this technique. METHODS: We report a prospective study of women with leiomyomata in excess of 10 cm in diameter who received GnRH agonist before magnetic-resonance-guided focused ultrasound treatment. Eligible participants were recruited from the gynecology outpatient clinics. Entry criteria were a minimal leiomyoma symptom severity score and confirmation of uterine dimensions based on screening magnetic resonance imaging. These women received a 3-month course of GnRH agonists followed by magnetic-resonance-guided focused ultrasound treatment. The primary outcome measurement was reported change in symptom severity score as judged by the Uterine Fibroid Symptom and Quality of Life questionnaire. Comparison was made at enrollment, treatment, and at 3, 6, and 12 months posttreatment. A secondary outcome was the measured change in target leiomyoma volume. RESULTS: Forty-nine women were enrolled in the study. There was a 45% reduction in median symptom severity score at 6 months and 48% at 12 months posttreatment, with 83% of women achieving at least a 10-point reduction in symptom scoring at 6 months and 89% at 12 months (P < .001). There was an average reduction in target leiomyoma volume of 21% overall at 6 months (P < .001) and 37% at 12 months (P < .001). No serious infective complications or emergency operative interventions were recorded. CONCLUSION: The use of GnRH agonist therapy before magnetic-resonance-guided focused ultrasound improves the thermoablative treatment effect. LEVEL OF EVIDENCE: II-3.

Magnetic resonance guided focused ultrasound surgery of uterine fibroids--the tissue effects of GnRH agonist pre-treatment.

OBJECTIVE: The purpose of this study was to determine the ablative effect of magnetic resonance guided focused ultrasound (MRgFUS) on fibroid tissue following the administration of gonadotrophin releasing hormone (GnRH) agonist. STUDY DESIGN: Fifty women with clinically symptomatic uterine fibroids were treated. Those with uterine diameter of 10 cm or greater were given 3 months pre-treatment with GnRH agonists. Data regarding number of ultrasound sonications, Joules of energy delivered and volume of thermal destruction was recorded. RESULTS: Twenty-seven subjects were given GnRH agonist therapy before MRgFUS and 23 women underwent MRgFUS without pre-treatment. All patients in both study groups completed MR guided FUS as an outpatient procedure with no device related adverse events reported. In the group of women who received GnRH agonists, the volume of ablation was significantly larger than that in the control group (0.06 cm3 versus 0.03 cm3, P<0.05), per Joule of energy applied. CONCLUSION: The use of GnRH agonists potentiates the thermal effects of MRgFUS in women undergoing treatment of uterine fibroids.

Human Sau3A repeated DNA is enriched in small polydisperse circular DNA from normal lymphocytes.

Representatives of the Sau3A family of short human repeated sequences [Meneveri et al., J. Mol. Biol. 186 (1985) 483-489] have been isolated from the small polydisperse circular DNA (spcDNA) of peripheral human lymphocytes. The prototype repeat is a 72-bp element which is at least partially tandemly repeated in spcDNA and human genomic DNA. In comparison with three major families of human repeated DNA, the Sau3A repeats are enriched in spcDNA. The function of spcDNA in normal and transformed eukaryotic cells is not understood and most studies have attempted to resolve this problem by molecular analysis of circular DNA isolated from cells in culture [see Rush and Misra, Plasmid 14 (1985) 177-191 for references]. We have studied the spcDNA present in normal uncultured human lymphocytes and present data pointing to the selective accumulation of the Sau3A family of repeated DNA within this population. The sequences of twelve of these repeats, the consensus sequence for this family and the sequence of a genomic repeat, are presented.

Sequence of the cell division gene CDC2 from Schizosaccharomyces pombe; patterns of splicing and homology to protein kinases.

The complete nucleotide sequence of a 2.9-kb DNA fragment containing the CDC2 gene-complementing activity from Schizosaccharomyces pombe has been determined. Within this region lies a 1.69-kb DNA sequence whose predicted amino acid sequence shows extensive homology to that previously deduced for the CDC28 gene product from Saccharomyces cerevisiae [Lörincz and Reed, Nature 307 (1984) 183-185]. Taken with the earlier observation that mutants in CDC2 can be rescued by the presence of the CDC28 gene [Beach, Durkacz and Nurse, Nature 300 (1982) 706-709], these results strongly suggest that the two genes code for similar functions. In contrast to the CDC28 gene, however, which contains no introns, the CDC2 coding sequence is split by four introns and from a comparison of the two sequences a consensus sequence for intron splicing in S. pombe can be established. Both CDC2 and CDC28 contain the consensus sequences for the ATP binding and phosphorylation acceptor sites of protein kinases such as bovine cAMP-dependent protein kinase (bov PK) and the src family of viral oncogene products.

An analysis of the genes encoding the 51.4- and 41.9-kDa toxins of Bacillus sphaericus 2297 by deletion mutagenesis: the construction of fusion proteins.

A series of deletion mutants have been constructed, in which varying numbers of amino acids have been deleted from both the N- and C-termini of both the 51.4- and 41.9-kDa toxins of Bacillus sphaericus. The results show that between 34-39 and 52-54 amino acids respectively at the N- and C-termini of the 51.4-kDa protein, are not essential for toxicity. In the case of the 41.9-kDa protein, the removal of only 7 amino acids from the C-terminus abolishes toxicity whilst at least 17 amino acids can be deleted from the N-terminus without loss of toxicity. A fusion protein with the 51.4-kDa derived sequence N-terminal to the 41.9-kDa sequence yielded a protein of Mr 87 kDa which was not toxic by itself. When supplemented with cells expressing only the 51.4-kDa protein, toxicity was restored. In contrast, another fusion protein, in which the gene order was reversed, was shown to be fully active in toxicity assays.

Small-scale techniques for the analysis of recombinant plasmids.

Using the cloning of part of the T-DNA of pTiC58 from Agrobacterium tumefaciens as an example, techniques are described which enable recombinant plasmids to be mapped and used as hybridization probes. In all cases the starting material is a colony of cells grown on an agar plate which is then subjected to lysis by lysozyme and Triton X-100 in volumes of the order of 300 microliters thus eliminating the need for handling and centrifuging liquid cultures under restrictive containment conditions.

Identification, cloning and sequence analysis of the Bacillus sphaericus 1593 41.9 kD larvicidal toxin gene.

A number of strains of the widespread aerobic soil bacterium, Bacillus sphaericus, possess crystalline inclusions of a toxin lethal to a variety of insect (larvae) which are vectors of major tropical diseases. Partial amino acid sequence data from one strain, B. sphaericus 2362 have permitted us to design oligonucleotide probes for identifying the toxin gene in the closely related B. sphaericus 1593. The gene was found to be contained within an EcoRI-HindIII fragment and was cloned in its entirety in the bacterial plasmid pUC12. The DNA sequence was determined together with the upstream and downstream controlling elements, and a sequence of 370 amino acids was deduced for the toxin protein. This is the first reported sequence of a B. sphaericus toxin gene and will facilitate further work in characterizing the genes from other strains of different virulence and host range. The data do not support the suggestion that the toxin is derived by proteolysis of a protoxin precursor.

Cytotoxicity and ADP-ribosylating activity of the mosquitocidal toxin from Bacillus sphaericus SSII-1: possible roles of the 27- and 70-kilodalton peptides.

Clones expressing regions of the 100-kDa Bacillus sphaericus SSII-1 mosquitocidal toxin (Mtx) as fusion proteins with glutathione S-transferase were constructed, and the toxin-derived peptides were purified. The in vitro ADP-ribosylation activities of these peptides and their effects on larvae and cells in culture were studied. Mtx25 (amino acids 30 to 493) was found to ADP-ribosylate two proteins with molecular masses of 38 and 42 kDa, respectively, in Culex quinquefasciatus (G7) cell extracts, in addition to ADP-ribosylating itself. Mtx21 (amino acids 30 to 870; or a combination of Mtx25 and Mtx26 (amino acids 259 to 870) caused mortality in C. quinquefasciatus larvae. Mtx25, Mtx26, or Mtx24 (amino acids 30 to 276) alone and Mtx24 in combination with Mtx26 were not toxic to larvae. Mtx21 and Mtx26 produced marked morphological changes in G7 cells and to a lesser extent in Aedes aegypti cells but had no effect on Anopheles gambiae or HeLa cells. Thus, a domain in the N-terminal region of the Mtx protein is sufficient for ADP-ribosylation of C. quinquefasciatus cell protein, and a domain in the C-terminal region is sufficient for toxicity to cultured C. quinquefasciatus cells; however, both regions are necessary for toxicity to mosquito larvae.