Recombinant carp parvalbumin, the major cross-reactive fish allergen: a tool for diagnosis and therapy of fish allergy

IgE-mediated reactions to fish allergens represent one of the most frequent causes of food allergy. We have constructed an expression cDNA library from carp (Cyprinus carpio) muscle in phage lambda gt11 and used serum IgE from a fish allergic patient to isolate 33 cDNA clones that coded for two parvalbumin isoforms (Cyp c 1.01 and Cyp c 1.02) with comparable IgE binding capacities. Both isoforms represented calcium-binding proteins that belonged to the beta-lineage of parvalbumins. The Cyp c 1.01 cDNA was overexpressed in Escherichia coli, and rCyp c 1.01 was purified to homogeneity. Circular dichroism analysis and mass spectroscopy showed that rCyp c 1.01 represented a folded protein with mainly alpha-helical secondary structure and a molecular mass of 11,416 Da, respectively. rCyp c 1.01 reacted with IgE from all fish-allergic patients tested (n = 60), induced specific and dose-dependent basophil histamine release, and contained most of the IgE epitopes (70%) present in natural allergen extracts from cod, tuna, and salmon. Therefore, it may be used to identify patients suffering from IgE-mediated fish allergy. The therapeutic potential of rCyp c 1.01 is indicated by our findings that rabbit Abs raised against rCyp c 1.01 inhibited the binding of IgE (n = 25) in fish-allergic patients to rCyp c 1.01 between 35 and 97% (84% mean inhibition) and that depletion of calcium strongly reduced IgE recognition of rCyp c 1.01. The latter results suggest that it will be possible to develop strategies for immunotherapy for fish allergy that are based on calcium-free hypoallergenic rCyp c 1.01 derivatives.     

Control of petal and pollen development by the plant cyclin-dependent kinase inhibitor ICK1 in transgenic <Emphasis Type="Italic">Brassica</Emphasis> plants

The cyclin-dependent protein kinases (CDKs) have a central role in cell cycle regulation and can be inhibited by the binding of small protein CDK inhibitors. The first plant CDK inhibitor gene ICK1 was previously identified in Arabidopsis thaliana. In comparison to known animal CDK inhibitors, ICK1 protein exhibits unique structural and functional properties. The expression of ICK1 directed by the constitutive CaMV 35S promoter was shown to inhibit cell division and plant growth. The aim of this study was to determine the effects of ICK1 overexpression on particular organs and cells. ICK1 was expressed in specific tissues or cells of Brassica napus L. plants using two tissue-specific promoters, Arabidopsis AP3 and Brassica Bgp1. Transgenic AP3-ICK1 plants were morphologically normal except for some modified flowers either without petals or with petals of reduced size. Surprisingly, petals of novel shapes such as tubular petals were also observed, indicating a profound effect of cell division inhibition on morphogenesis. The cell size in the smaller modified petals was similar to that in control petals, suggesting that the reduction of petal size is mainly due to the reduction of cell numbers and that the inhibition of cell division does not necessarily lead to an increase in cell size. Transgenic Bgp1-ICK1 plants were normal morphologically; however, dramatic decreases in seed production were observed in some plants. In those plants, the ability of pollen to germinate and pollen nuclear number were affected. These results are discussed in relation to the cell cycle and plant development.     

A bacterial artificial chromosome-based genetic linkage map of the nematode Pristionchus pacificus

To understand the evolution of developmental processes, nonmodel organisms in the nematodes, insects, and vertebrates are compared with established model systems. Often, these comparisons suffer from the inability to apply sophisticated technologies to these nonmodel species. In the nematode Pristionchus pacificus, cellular and genetic analyses are used to compare vulva development to that of Caenorhabditis elegans. However, substantial changes in gene function between P. pacificus and C. elegans limit the use of candidate gene approaches in studying P. pacificus mutations. To facilitate map-based cloning of mutations in P. pacificus, we constructed a BAC-based genetic linkage map. A BAC library of 13,440 clones was generated and completely end sequenced. By comparing BAC end and EST sequences between the "wild-type" strain P. pacificus var. California and the polymorphic strain P. pacificus var. Washington, 133 single-stranded conformational polymorphisms were identified. These markers were tested on a meiotic mapping panel of 46 randomly picked F(2) animals after a cross of the two strains, providing the first genetic linkage map of P. pacificus. A mapping strategy using two selected markers per chromosome was devised and the efficiency of this approach was illustrated by the mapping of the Ppa-unc-1/Twitchin gene.     

Exogenous nitric oxide triggers Neospora caninum tachyzoite-to-bradyzoite stage conversion in murine epidermal keratinocyte cell cultures

Neospora caninum, like Toxoplasma gondii, undergoes stage conversion in chronically infected animals, and forms tissue cysts which contain the slowly proliferating bradyzoite stage. These tissue cysts are delineated by a cyst wall, protect the parasite from physiological and immunological reactions on part of the host, and bradyzoites remain viable within an infected host for many years. However, unlike T. gondii, N. caninum bradyzoites have been difficult to obtain using in vitro culture techniques, and current protocols, based on those developed for T. gondii, have been shown to be not very efficient in promoting tachyzoite-to-bradyzoite stage conversion. We report here an alternative in vitro culture method to obtain stage conversion of N. caninum from the proliferative to the cystic stage by using the Nc-Liverpool isolate, murine epidermal keratinocytes as host cells, and continuous treatment of infected cultures with 70 microM sodium nitroprusside for up to 8 days. This treatment significantly reduced parasite proliferation as assessed by Neospora-specific quantitative real-time PCR. The expression of bradyzoite markers was analysed by immunofluorescence following 4 and 8 days of in vitro culture using antibodies directed against bradyzoite antigen 1, the mAbCC2, and the lectin Dolichos biflorus agglutinin. Expression of the tachyzoite-specific immunodominant antigen NcSAG1 and the tachyzoite antigen NcMIC1 was also assessed. Transmission electron microscopy revealed that the majority of parasitophorous vacuoles were in the process of forming a distinct cyst wall through accumulation of granular material at the periphery of the vacuole, and parasites exhibited the typical features of bradyzoites. These findings demonstrate the usefulness of this culture technique as a promising way to study tachyzoite-to-bradyzoite stage conversion in N. caninum in vitro.     

Heterosis and outbreeding depression in descendants of natural immigrants to an inbred population of song sparrows (Melospiza melodia)

We studied heterosis and outbreeding depression among immigrants and their descendants in a population of song sparrows on Mandarte Island, Canada. Using data spanning 19 generations, we compared survival, seasonal reproductive success, and lifetime reproductive success of immigrants, natives (birds with resident-hatched parents and grandparents), and their offspring (F1s, birds with an immigrant and a native parent, and F2s, birds with an immigrant grandparent and resident-hatched grandparent in each of their maternal and paternal lines). Lifetime reproductive success of immigrants was no worse than that of natives, but other measures of performance differed in several ways. Immigrant females laid later and showed a tendency to lay fewer clutches, but had relatively high success raising offspring per egg produced. The few immigrant males survived well but were less likely to breed than native males of the same age that were alive in the same year. Female F1s laid earlier than expected based on the average for immigrant and native females, and adult male F1s were more likely to breed than expected based on the average for immigrant and native males. The performance differences between immigrant and native females and between F1s and the average of immigrants and natives are consistent with the hypothesis that immigrants were disadvantaged by a lack of site experience and that immigrant offspring benefited from heterosis. However, we could not exclude the possibility that immigrants had a different strategy for optimizing reproductive success or that they experienced ecological compensation for life-history parameters. For example, the offspring of immigrants may have survived well because immigrants laid later and produced fewer clutches, thereby raising offspring during a period of milder climatic conditions. Although sample sizes were small, we found large performance differences between F1s and F2s, which suggested that either heterosis was associated with epistasis in F1s, that F2s experienced outbreeding depression, or that both phenomena occurred. These findings indicate that the performance of dispersers may be affected more by fine-scale genetic differentiation than previously assumed in this and comparable systems.     

A colorimetric assay for the quantitation of free adenine applied to determine the enzymatic activity of ribosome-inactivating proteins

Adenine quantitation is required for a variety of applications. To date, the prevalent method for quantifying free adenine, in a variety of applications, is the detection of fluorescent-derivatized adenine by HPLC. For the present study, we developed a high-throughput, nonradioactive, enzyme-based colorimetric adenine quantitation assay that is performed in one multireaction incubation step. The assay does not require adenine derivatization and is designed for microplates. The key step is the conversion of adenine to adenosine monophosphate by adenine phosphoribosyl transferase. Subsequent reactions finally produce three inorganic phosphate ions per adenine molecule. Phosphate is quantitated by the color-generating phosphorylysis of a particular purine derivate. Ribosome-inactivating proteins that release adenine from polynucleotides are often used to investigate intracellular protein trafficking and are important for the design of immunotoxins. We therefore used ricin, dianthin, saporin, and a variety of saporin fusion proteins to show that this method is suitable for quantifying adenine release using different substrates. The measured rate of adenine release and substrate specificity are comparable to those determined by HPLC and radioactive detection techniques.     

Measurement of resiniferatoxin in cerebrospinal fluid by high-performance liquid chromatography

A sensitive and simple high-performance liquid chromatographic (HPLC) assay was developed for the quantification of resiniferatoxin (RTX) in canine cerebrospinal fluid (CSF). A reversed-phase C(18) column and acetonitrile in 0.02 M NaH(2)PO(4) as mobile phase provided satisfactory resolution for RTX analysis. Direct HPLC analysis of the CSF samples without sample extraction or preparation improves the accuracy and detection limits of this assay. This assay was applied to measure CSF RTX content to test this method for research and clinical applications related to studies examining its analgesia effects.     

Homology-Dependent Maternal Inhibition of Developmental Excision of Internal Eliminated Sequences in Paramecium tetraurelia

Thousands of single-copy internal eliminated sequences (IESs) are excised from the germ line genome of ciliates during development of the polygenomic somatic macronucleus, following sexual events. Paramecium IESs are short, noncoding elements that frequently interrupt coding sequences. No absolutely conserved sequence element, other than flanking 5′-TA-3′ direct repeats, has been identified among sequenced IESs; the mechanisms of their specific recognition and precise elimination are unknown. Previous work has revealed the existence of an epigenetic control of excision. It was shown that the presence of one IES in the vegetative macronucleus results in a specific inhibition of the excision of the same element during the development of a new macronucleus, in the following sexual generation. We have assessed the generality and sequence specificity of this transnuclear maternal control by studying the effects of macronuclear transformation with 13 different IESs. We show that at least five of them can be maintained in the new macronuclear genome; sequence specificity is complete both between genes and between different IESs in the same gene. In all cases, the degree of excision inhibition correlates with the copy number of the maternal IES, but each IES shows a characteristic inhibition efficiency. Short internal IES-like segments were found to be excised from two of the IESs when excision between normal boundaries was inhibited. Available data suggest that the sequence specificity of these maternal effects is mediated by pairing interactions between homologous nucleic acids.