Comparison of three variations of the radioallergosorbent test-inhibition assay for measuring allergenic activity of grass pollen extracts

Three variations of the radioallergosorbent (RAST)-inhibition assay (RI) have been compared for measuring the allergenic activity of grass pollen extracts. The main difference between the three assays consisted in the solid supports to which the allergens were coupled. These supports were paper disks, microcrystalline cellulose, and Matrex. It could be demonstrated that all three variations of RI yielded almost identical results as expressed by their F value (F_o). Correlations between the three methods were highly significant (P < 0.001). Three independent valid assays showed an excellent reproducibility of each test system (P < 0.01). Three different preparations of each of the supports yielded highly reproducible F_o (0.76 ± 3% SD). Evaluation of the 50% inhibition values achieved with the different assays based on protein showed differences between the three supports. Provided the same allergen extract is coupled to the different solid supports and the results are related to a reference preparation, different laboratories using different forms of RI will be able to compare their results.     

Geochemical study of vertebrate fossils from the Upper Cretaceous (Santonian) Csehbánya Formation (Hungary): Evidence for a freshwater habitat of mosasaurs and pycnodont fish

The diverse vertebrate remains from the Upper Cretaceous freshwater settings at Iharkút, Hungary, contain two fossil groups, Pycnodontiformes fish and Mosasauridae that are almost exclusively known from marine palaeo-environments. Hence, their appearance in alluvial sediments is very unusual. Trace element and isotope compositions of the remains have been analyzed to investigate the taphonomy and the ecological differences among the different fossil groups present at Iharkút.All examined fossils have undergone post-depositional diagenetic alteration, which resulted in high concentrations of REE, U, and Fe, together with almost complete homogenization of δ¹⁸O_CO3 values. Similar REE patterns in different fossils suggest a common origin for all remains, hence the discovered species most likely lived in the same local ecosystem. Despite partial diagenetic overprinting, the δ¹⁸O_PO4 values of the fossils indicate sufficient taxon-specific isotopic diversity to permit some broad conclusions on the palaeo-environment of the fossils. In particular, it is apparent that the isotopic composition of the Pycnodontiformes fish and Mosasauridae remains is most compatible with a freshwater palaeo-habitat and incompatible with a marine palaeo-environment. In addition, the Sr concentration and isotope data indicate that the Pycnodontiformes and Mosasauridae likely lived predominantly in a freshwater environment and were not simply occasional visitors to the Iharkút river ecosystem.Regarding other fossil groups, high δ¹⁸O_PO4 values of Alligatoroidea and Iharkutosuchus teeth suggest that these small crocodile species might have inhabited swamps and ponds where the water was relatively rich in ¹⁸O due to evaporation.     

Spatial pattern of cauliflower mosaic virus 35S promoter-luciferase expression in transgenic hybrid aspen trees monitored by enzymatic assay and non-destructive imaging

A protocol has been developed for efficiently transforming and regenerating the hybrid aspenPopulus tremula x P. tremuloides. Stem segments were co-cultivated with a strain ofAgrobacterium tumefaciens carrying a disarmed binary vector conferring resistance to kanamycin or hygromycin. The respective vectors also carried a fused bacterialluxF2 gene expressed from the cauliflower mosaic virus 35S promoter. All transformants had a normal phenotype. Genetic tranformation and stable integration of the heterologous DNA was confirmed by Southern hybridization and luciferase expression. The latter was measured by destructive enzymatic assay throughout the transformatnt and by non-destructive image analysis in leaves left attached to intact plants. Both measurement techniques detected marked within- and between-organ variation in luciferase expression. However, the spatial patterns detected by each technique in the leaves were similar. The results indicate thatin vivo imaging of light emission can be used to measure repeatedly the expression of a promoter-luciferase gene fusion in a particular leaf over an extended time period. It was also demonstrated that enzymatically assayed luciferase activity in leaves was notably lowere in transgenic hybrid aspen plants than in tobacco plants transformed with the same vector. This was not due to a difference in luciferase enzyme activity between the two species, and therefore indicated that the 35S promoter is not as active in hybrid aspen as in tobacco.     

Revision of the amino acid sequence of human heart fatty acid-binding protein

Summary Cardiac-type fatty acid-binding protein (cFABP) from human heart muscle of three individuals was isolated and characterized as pI 5.3-cFABP. The proteins were structurally analyzed by tryptic peptide mapping, application of plasma desorption time-of-flight mass spectrometry and amino acid sequencing. All three preparations of human heart FABP, having 132 amino acids, differed from the published sequence [Offner et al. Biochem J 251: 191–198, 1988] in position 104, where Leu is found instead of Lys, and in position 124, where Cys is found instead of Ser.     

Cofractionation of hela cell replication proteins with Ors-binding activity

Ors (origin enriched sequence) 8 is a mammalian autonomously replicating DNA sequence previously isolated by extrusion of nascent monkey (CV-1) DNA in early S phase. A 186 bp fragment of ors 8 has been identified as the minimal sequence required for origin function, since upon its deletion the in vivo and in vitro replication activity of this ors is abolished. We have fractionated total HeLa cell extracts on a DEAE-Sephadex and then on a Affi-Gel Heparin column and identified a protein fraction that interacts with the 186 bp fragment of ors 8 in a specific manner. The same fraction is able to support the in vitro replication of ors 8 plasmid. The ors binding activity (OBA) present in this fraction sediments at approximately 150 kDa in a glycerol gradient. Band-shift elution experiments of the specific protein-DNA complex detect by silver-staining predominantly two protein bands with molecular weights of 146 kDa and 154 kDa, respectively. The fraction containing the OBA is also enriched for polymerases α and δ, topoisomerase II, and replication protein A, (RP-A).     

Effects of recombinant human colony stimulating factors (CSF) (granulocyte-macrophage CSF, granulocyte CSF, and CSF-1) on human monocyte/macrophage differentiation

Purified recombinant human granulocyte-macrophage (rhuGM)-CSF, rhuG-CSF, and rhuCSF-1 were evaluated for their capacity to influence the differentiation of U-937 cells and normal human monocytes. The human U-937 cell line represents an early stage of monocytic differentiation. It was found that rhuGM-CSF and rhuG-CSF, but not rhuCSF-1, induced phenotypic changes consistent with monocyte/macrophage differentiation in U-937 cells. After 3 days of culture in the presence of either rhuGM-CSF or rhuG-CSF, a small but significant proportion of U-937 cells were able to reduce nitroblue tetrazolium. Nitroblue tetrazolium reduction, however, was maximally induced when rhuGM-CSF and rhuG-CSF were added in combination. These changes were accompanied by increased alpha-naphthyl acetate esterase activity, acquisition of macrophage morphology, Mo-1 Ag expression, and decreased cell proliferation. rhuGM-CSF alone also induced expression of the c-fms proto-oncogene (CSF-1 receptor) in U-937 cells and this expression was enhanced by the combination of rhuGM-CSF and rhuG-CSF. In cultured normal human peripheral blood monocytes, representing a late stage of maturation, rhuGM-CSF and rhuCSF-1 differentially increased Mo-1 and My-4 Ag expression, respectively, whereas rhuG-CSF was without effect. Our results suggest that the interaction of GM-CSF, G-CSF, and CSF-1 may play a fundamental role in the early and late stages of the human monocyte/macrophage differentiation process.     

Growth of Wolinella succinogenes with elemental sulfur in the absence of polysulfide

Wolinella succinogenes grows by anaerobic respiration with formate and polysulfide. Polysulfide forms spontaneously from sulfur and sulfide. Here we report that this eubacterium also grows with formate and elemental sulfur under conditions that do not allow polysulfide formation. With the appropriate amount of Fe²⁺ added to the medium, the concentration of polysulfide was calculated to be 0.4 nM, which is 1/400th of the concentration that of dissolved elemental sulfur. At commensurable growth rates, the growth yield with sulfur was one quarter of that with polysulfide as electron acceptor. The same low growth yield either with sulfur or with polysulfide as electron acceptor was measured for a Δpsr mutant that lacks the genes encoding polysulfide reductase (Psr). Received: 8 June 1995 / Accepted: 12 September 1995