Specific V beta T cell subsets are associated with cat and birch pollen allergy in humans.

Cognate interaction between TCRs and MHC class II molecules plays an important role in initiating the allergen-specific immune response. Therefore, we analyzed the TCR distribution of human PBLs of 56 atopic and nonatopic (NA) individuals, including 4 monozygotic twin pairs, from two extended and four nuclear families. The expression of 23 V beta and 3 V alpha elements was analyzed. The blood samples of symptomatic birch pollen-sensitized individuals that were taken < or = 6 wk after the birch pollen season (n = 8) showed a significantly higher frequency of V beta 16.1+ and V beta 20.1+ T cells compared with the blood samples of birch pollen-sensitized individuals that were obtained out of allergen season (n = 10) or from NA individuals (p < 0.0005 and p < 0.0001, respectively). Allergen-specific lymphocyte proliferation was detected in the allergic individuals, and the distribution of V beta 16.1+ and V beta 20.1+ T cells returned to normal levels after the pollen season. The frequency of these V beta-expressing T cells correlated with the levels of allergen-specific IgE Abs. In addition, cat-sensitized individuals (n = 8) showed a significantly higher frequency of V beta 17.1-expressing T cells than did NA individuals (p < 0.005). Our results indicate restricted TCR-V beta gene usage in cat and birch pollen allergies; we suggest that both genetic and environmental factors contribute to TCR-V beta gene expression and to the development of a specific T cell response.

Conservation of mitochondrial targeting sequence function in mitochondrial and hydrogenosomal proteins from the early-branching eukaryotes Crithidia, Trypanosoma and Trichomonas.

Kinetoplastid protozoa are the earliest-branching eukaryotes to possess a true mitochondrion. This organelle is host to a variety of intriguing and unique features, including RNA editing. We examined the characteristics of protein import into mitochondria of Trypanosoma brucei. Dihydrofolate reductase (DHFR) carrying a yeast mitochondrial targeting signal was correctly translocated into trypanosome mitochondria in vivo, as were DHFR fusion proteins bearing two unusually short (7-9 amino acids) presequences from trypanosomatids. The short trypanosomal targeting signals were functional in Saccharomyces cerevisiae as well, but their targeting efficiency was lower and processing was absent. Trichomonads branched even earlier than kinetoplastids in eukaryotic evolution and contain energy-generating organelles called hydrogenosomes. The origin of hydrogenosomes has been controversial, but most evidence suggests that they are related to mitochondria. Putative hydrogenosomal targeting signals from Trichomonas vaginalis are short (5-12 amino acids). Three such sequences were capable of targeting a passenger protein to mitochondria both in yeast and in trypanosomes, and one of the hydrogenosomal presequences was efficiently processed in both organisms. These findings suggest a resemblance between the import machineries of mitochondria and hydrogenosomes.

In vitro import of proteins into mitochondria of Trypanosoma brucei and Leishmania tarentolae.

In eukaryotic evolution, the earliest branch of organisms to have mitochondria are the trypanosomatids. Their mitochondrial biogenesis not only includes import of most proteins, but also, unlike in other organisms, import of the whole set of tRNAs. In order to investigate these processes, we devised novel procedures for the isolation of mitochondria from two trypanosomatid species: Trypanosoma brucei and Leishmania tarentolae. Isotonic cell lysis followed by equilibrium density centrifugation in Nycodenz gradients yielded mitochondrial fractions exhibiting a membrane potential. Furthermore, we have used these fractions to reconstitute import of mitochondrial matrix proteins in vitro. Energy-dependent uptake of an artificial precursor protein, containing a trypanosomal presequence attached to mouse dihydrofolate reductase and of yeast mitochondrial alcohol dehydrogenase could be demonstrated. The presequences of both proteins were processed in T. brucei whereas only the trypanosomal one was cleaved in L. tarentolae. Trypsin pretreatment abolished the ability of the mitochondria to import proteins, indicating the involvement of proteinaceous components at the surface of mitochondria.

Post-transcriptional control of hsp70 mRNA in Trypanosoma brucei.

The control of hsp70 mRNA levels was investigated using transgenic bloodstream and procyclic trypanosomes. Heat shock of procyclic and bloodstream trypanosomes caused no significant change in overall protein synthesis, but led to a 2-3-fold increase in the relative hsp70 mRNA level in bloodstream trypanosomes. Incubation of procyclic trypanosomes at 35 degrees C for up to 18 h increased the level of hsp70 mRNA only marginally. The expression of actin and hsp70 mRNAs was markedly reduced in late log phase procyclic trypanosomes but PARP mRNA levels remained constant. Measurements of phleomycin-binding-protein RNAs bearing 3'- and 5'-untranslated regions from the actin, PARP or hsp70 loci indicated that both the heat-shock and cell-density effects were mediated by the untranslated regions. No significant promoter activity was detected in the different hsp70 locus intergenic regions in transient assays.

Import of a DHFR hybrid protein into glycosomes in vivo is not inhibited by the folate-analogue aminopterin.

Dihydrofolate reductase fusion proteins have been widely used to study conformational properties of polypeptides translocated across membranes. We have studied the import of dihydrofolate reductase fusion proteins into glycosomes and mitochondria of Trypanosoma brucei. As signal sequences we used the last 22 carboxy-terminal amino acids of glycosomal phosphoglycerate kinase for glycosomes, and the cleavable presequences of yeast cytochrome b2 or cytochrome oxidase subunit IV for mitochondria. Upon addition of aminopterin, a folate analogue that stabilizes the dihydrofolate reductase moiety, import of the fusion protein targeted to glycosomes was not inhibited, although the results of protease protection assays showed that the fusion protein could bind the drug. Under the same conditions, import of a DHFR fusion protein targeted to mitochondria was inhibited by aminopterin. When DHFR fusion proteins targeted simultaneously to both glycosomes and mitochondria were expressed, import into mitochondria was inhibited by aminopterin, whereas uptake of the same proteins into glycosomes was either unaffected or slightly increased. These findings suggest that the glycosomes possess either a strong unfolding activity or an unusually large or flexible translocation channel.

Protein trafficking in kinetoplastid protozoa.

The kinetoplastid protozoa infect hosts ranging from invertebrates to plants and mammals, causing diseases of medical and economic importance. They are the earliest-branching organisms in eucaryotic evolution to have either mitochondria or peroxisome-like microbodies. Investigation of their protein trafficking enables us to identify characteristics that have been conserved throughout eucaryotic evolution and also reveals how far variations, or alternative mechanisms, are possible. Protein trafficking in kinetoplastids is in many respects similar to that in higher eucaryotes, including mammals and yeasts. Differences in signal sequence specificities exist, however, for all subcellular locations so far examined in detail--microbodies, mitochondria, and endoplasmic reticulum--with signals being more degenerate, or shorter, than those of their higher eucaryotic counterparts. Some components of the normal array of trafficking mechanisms may be missing in most (if not all) kinetoplastids: examples are clathrin-coated vesicles, recycling receptors, and mannose 6-phosphate-mediated lysosomal targeting. Other aspects and structures are unique to the kinetoplastids or are as yet unexplained. Some of these peculiarities may eventually prove to be weak points that can be used as targets for chemotherapy; others may turn out to be much more widespread than currently suspected.

New adhesives in Class V restorations under combined load and simulated dentinal fluid.

OBJECTIVES: This study compared the efficacy of three dentinal adhesives using the "all etch" technique (All-Bond 2, Bisco; Scotchbond MP,3M Dental Products Co.; OptiBond, Kerr) with a dentinal adhesive which still uses phosphoric acid to condition enamel and a self-etching primer for dentin (A.R.T.-Bond, Coltene/Whaledent). METHODS: Eight V-shaped mixed Class V restorations were placed per group in extracted human premolars. The restorations were subjected to 1,200,000 mechanical occlusal cycles (max. force 49 N; frequency 1.7 Hz) and 3,000 simultaneous thermal cycles (5-50-5 degrees C). Dentinal fluid was simulated using 1:3 diluted horse serum and fed into the pulp chamber both during restoration and loading. Percentages of "continuous margin" were assessed on SEM replicas of enamel and dentinal margins at 200x magnification immediately before and after stressing, respectively. RESULTS: No significant differences were observed before stress between the materials either in enamel or in dentin. After stress, however, OptiBond and A.R.T.-Bond performed significantly better in dentin than the two other adhesives (Kruskal-Wallis, Mann-Whitney; p < 0.05). Although high initial values were observed, All-Bond 2 and Scotchbond MP were not stress-resistant under simulated physiological conditions. SIGNIFICANCE: The predicted clinical potential of All-Bond 2 and Scotchbond MP is inferior to that of OptiBond and A.R.T.-Bond.

[Composite polymers--an amalgam substitute for deciduous tooth cavities?]. / Kompomere--Amalgamersatz für Milchzahnkavitäten?

In this prospective clinical study, 29 class II restorations in deciduous teeth were placed in 17 patients using the recently developed compomer Dyract. The restorations were clinically and macrophotographically evaluated immediately after placement and after 6 months in situ. In addition, marginal adaptation and loss of substance were quantitated using replicas. Clinical and macrophotographical findings were excellent. Quantitative marginal analysis scored 92.5% "continuous margin" immediately after placement of the restorations and 95.4% after 6 months. Mean loss of substance at the margins of the restorations was 20.8 microns with a standard deviation of 64.6 microns. Providing that the positive results of this short term study are confirmed by long term data, Dyract may replace amalgam in deciduous teeth.