Rational design of a hypoallergenic Phl p 7 variant for immunotherapy of polcalcin-sensitized patients.

Polcalcins are important respiratory panallergens, whose IgE-binding capacity depends on the presence of calcium. Since specific immunotherapy is not yet available for the treatment of polcalcin-sensitized patients, we aimed to develop a molecule for efficient and safe immunotherapy. We generated a hypoallergenic variant of the grass pollen polcalcin Phl p 7 by introducing specific point mutations into the allergen's calcium-binding regions. We thereby followed a mutation strategy that had previously resulted in a hypoallergenic mutant of a calcium-binding food allergen, the major fish allergen parvalbumin. Dot blot assays performed with sera from Phl p 7-sensitized patients showed a drastically reduced IgE reactivity of the Phl p 7 mutant in comparison to wildtype Phl p 7, and basophil activation assays indicated a significantly reduced allergenic activity. Rabbit IgG directed against mutant rPhl p 7 blocked patients' IgE binding to wildtype Phl p 7, indicating the mutant's potential applicability for immunotherapy. Mass spectrometry and circular dichroism experiments showed that the mutant had lost the calcium-binding capacity, but still represented a folded protein. In silico analyses revealed that the hypoallergenicity might be due to fewer negative charges on the molecule's surface and an increased molecular flexibility. We thus generated a hypoallergenic Phl p 7 variant that could be used for immunotherapy of polcalcin-sensitized individuals.

Concentration of free amino acids in human milk of women with gestational diabetes mellitus and healthy women.

OBJECTIVE: It is generally agreed that breastfeeding has a positive effect on the metabolic situation in diabetic mothers. However, negative long-term effects are described for breastfed offspring of diabetic women. It is unknown if the composition of free amino acids (FAAs) in breastmilk of women with gestational diabetes mellitus (GDM) differs from that in milk of healthy women. We studied the amount of FAAs in breastmilk of women with GDM and women with normal glucose tolerance (NGT). SUBJECTS AND METHODS: Human milk samples of 68 women (21 GDM and 47 NGT) were analyzed. Contents of FAAs in milk samples, obtained within the first 4 days after delivery (colostrum) and 6 weeks later (mature milk), were analyzed using high-performance liquid chromatography. Total amounts of FAAs in colostrum and in mature milk were compared between the groups. The impact of maternal age, body mass index (BMI), gestational age at birth, birth weight, and diagnosis of GDM on the total amount of FAAs was evaluated. RESULTS: Overall, the total amount of FAAs increased significantly from colostrum to mature milk in both groups (p<0.001). The total amount of FAAs did not significantly differ between GDM and NGT in colostrum and in mature milk (1,560 µmol/L vs. 1,730 µmol/L and 2,440 µmol/L vs. 2,723 µmol/L, respectively). No significant influence on the total amount of FAAs at both measurements of maternal age, BMI, gestational age at birth, birth weight, and diagnosis of GDM could be observed by regression analyses. CONCLUSION: The content of FAAs of human milk does not significantly differ between women with GDM and women with NGT.

Cu2+-cyclen as probe to identify conformational states in guanine nucleotide binding proteins.

(31)P NMR spectroscopy is a suitable method for identifying conformational states in the active site of guanine nucleotide binding proteins detecting the nucleotide placed there. Because there is no labeling necessary, this method is gaining increasing interest. By (31)P NMR spectroscopy two major conformational states, namely state 1(T) and state 2(T), can be detected in active Ras protein characterized by different chemical shifts. Depending on the conformational state Ras shows clearly different physiological properties. Meanwhile analogous conformational equilibria could also be shown for other members of the Ras superfamily. It is often difficult to determine the conformational states of the proteins on the basis of chemical shift alone; therefore, direct detection would be a great advantage. With the use of Cu(2+)-cyclen which selectively interacts only with one of the major conformational states (state 1) one has a probe to distinguish between the two states, because only proteins existing in conformational state 1 interact with the Cu(2+)-cyclen at low millimolar concentrations. The suitability was proven using Ras(wt) and Ras mutants, Ras complexed with GTP, GppNHp, or GTPγS, as well as two further members of the Ras superfamily namely Arf1 and Ran.

Survey of pedestrian detection for advanced driver assistance systems.

Advanced driver assistance systems (ADASs), and particularly pedestrian protection systems (PPSs), have become an active research area aimed at improving traffic safety. The major challenge of PPSs is the development of reliable on-board pedestrian detection systems. Due to the varying appearance of pedestrians (e.g., different clothes, changing size, aspect ratio, and dynamic shape) and the unstructured environment, it is very difficult to cope with the demanded robustness of this kind of system. Two problems arising in this research area are the lack of public benchmarks and the difficulty in reproducing many of the proposed methods, which makes it difficult to compare the approaches. As a result, surveying the literature by enumerating the proposals one--after-another is not the most useful way to provide a comparative point of view. Accordingly, we present a more convenient strategy to survey the different approaches. We divide the problem of detecting pedestrians from images into different processing steps, each with attached responsibilities. Then, the different proposed methods are analyzed and classified with respect to each processing stage, favoring a comparative viewpoint. Finally, discussion of the important topics is presented, putting special emphasis on the future needs and challenges.

Absence of the JAK2 mutation V617F in CD34+ hematopoietic stem and progenitor cells from patients with BCR-ABL-positive CML in chronic phase and blast crisis.

In this study, we examined highly enriched CD34+ cells from patients with BCR-ABL-positve CML in chronic phase or blast crisis for the JAK2 V617F activation mutation by sequencing. The cells examined did not bear the mutation irrespective of the disease stage. This finding suggests that the previously described increase of expression and kinase activity of JAK2 in CML cells does not result from the JAK2 V617F activation mutation and that transformation into blast crisis is not associated with the occurrence of this mutation.

A novel mechanism for the modulation of the Ras-effector interaction by small molecules.

When proteins require different conformations for their biological function, all these functional states have to coexist simultaneously in solution. However, the corresponding Gibbs free energy differences are usually rather high and thus the conformation with lowest energy predominates in solution whereas the populations of the states with higher energy (excited states) are very small. A stabilization of these excited states can be used as a novel principle to influence the activity of proteins by small molecules. For a proof of this principle, we selected the Ras protein that was shown by (31)P NMR spectroscopy to exist in solution in at least two different conformational states in its GTP form. One of these states shows a drastically reduced affinity to effectors. With Zn(2+)-cyclen we found a small molecule which selectively stabilizes the weak-binding state. It may serve as lead compound for the development of a new type of Ras-inhibitors.

Distinct molecular phenotype of malignant CD34(+) hematopoietic stem and progenitor cells in chronic myelogenous leukemia.

Chronic myelogenous leukemia (CML) is a malignant disorder of the hematopoietic stem cell characterized by the BCR-ABL oncogene. We examined gene expression profiles of highly enriched CD34(+) hematopoietic stem and progenitor cells from patients with CML in chronic phase using cDNA arrays covering 1.185 genes. Comparing CML CD34(+) cells with normal CD34(+) cells, we found 158 genes which were significantly differentially expressed. Gene expression patterns reflected BCR-ABL-induced functional alterations such as increased cell-cycle and proteasome activity. Detoxification enzymes and DNA repair proteins were downregulated in CML CD34(+) cells, which might contribute to genetic instability. Decreased expression of junction plakoglobulin and CXC chemokine receptor 4 (CXCR-4) might facilitate the release of immature precursors from bone marrow in CML. GATA-2 was upregulated in CML CD34(+) cells, suggesting an increased self-renewal in comparison with normal CD34(+) cells. Moreover, we found upregulation of the proto-oncogene SKI and of receptors for neuromediators such as opioid mu1 receptor, GABA B receptor, adenosine A1 receptor, orexin 1 and 2 receptors and corticotropine-releasing hormone receptor. Treatment of CML progenitor cells with the selective adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) resulted in a dose-dependent significant inhibition of clonogenic growth by 40% at a concentration of 10(-5) M, which could be reversed by the equimolar addition of the receptor agonist 2-chloro-N6-cyclopentyladenosine (P<0.05). The incubation of normal progenitor cells with DPCPX resulted in an inhibition of clonogenic growth to a significantly lesser extent in comparison with CML cells (P<0.05), suggesting that the adenosine A1 receptor is of functional relevance in CML hematopoietic progenitor cells.

Expression of the tyrosine kinase c-kit is an independent prognostic factor in patients with small cell lung cancer.

In a retrospective analysis of 203 patients with small cell lung cancer (SCLC), we examined the prognostic value of c-kit expression on survival. Expression of c-kit was examined immunohistochemically in formalin-fixed, paraffin-embedded tissue sections. c-kit was observed in 87.7% of SCLC tumors. Using the Kaplan-Meier model, we found that lack of c-kit expression was associated with significantly shorter survival time compared to the presence of c-kit expression (mean survival 151 +/- 27 vs. 358 +/- 49 days, p = 0.0084). Moreover, the proportion of c-kit(+) cells within the tumor was also related to survival time. Patients with tumors in which >75% of cells stained positive for c-kit had a mean overall survival time of 424 (+/-72) compared to 295 (+/-67) days for patients with 25-75% c-kit(+) tumor cells. Patients with tumors containing <25% c-kit(+) cells had the worst survival, with 164 (+/-24) days (p = 0.0033). Further parameters associated with short survival times were low performance status, elevated levels of lactate dehydrogenase and higher stage according to the TNM classification. Multivariate analysis using the Cox regression model showed that the proportion of c-kit(+) cells within the tumor specimen was one of 3 independent prognostic parameters (p = 0.004) for overall survival next to TNM classification (p = 0.001) and performance status (p < 0.001).

Inhibition of angiogenesis in vitro by alphav integrin-directed antisense oligonucleotides.

The integrin alpha v beta 3 plays a central role in angiogenesis. In this study, we used antisense oligodeoxyribonucleotides (ONs) directed against the alpha v subunit of alpha v beta 3 to inhibit integrin expression. Ten ON sequences, which were selected by systematic alignment of computer-predicted secondary structures of alpha v mRNA, were transfected into human umbilical vein endothelial cells (HUVECs). Following stimulation by PMA, five antisense ONs significantly inhibited alpha v mRNA and protein expression in activated HUVEC at a concentration of 0.05 mciroM with complete prevention of PMA-induced alpha v up-regulation by the most potent antisense ON. Inhibition of alpha v expression was associated with significant inhibition of migration of HUVEC by 28% and had no effect on proliferation and apoptosis. Moreover, transfection of antisense ON inhibited the formation of tube-like structures of HUVEC in Matrigel by 44%. In a cell culture model of angiogenesis consisting of a co-culture of endothelial cells with fibroblasts, transfection of antisense ONs resulted in an inhibition of tube formation of 61%. In conclusion, alpha v antisense ONs are potent inhibitors of angiogenesis in vitro. They might, therefore, be a therapeutic alternative to antagonists, which directly bind to alpha v integrins, and might be useful for the treatment of malignant tumors and hematological malignancies.