Protective effect of moderate overweight on bone density of the hip joint in elderly and old Austrians

The effect of weight, classified by body mass index (BMI), on bone mass (BMC) of the whole body and on bone mineral density BMD of the hip joint was analysed in a sample of 120 Austrians of Vienna and surroundings. The 68 females and 52 males of this cross sectional study ranged in age between 60 and 92 years (x = 71.7 +/- 7.7). Age distribution was not significantly different between sexes. The WHO (1997) classification of body mass index (BMI) was used for weight classification, i.e. normal weight (BMI 18.5-24.99) and moderate overweight (BMI 25.0-29.99). Obese subjects (BMI 30+) were not included in this study. Bone mass of the whole body as well as bone density of the hip joint were determined by Dual-energy-X-ray absorptiometry (DEXA) using a hologic 2000 scanner. As expected BMC and BMD values were significantly higher in males than in females. While in both females and males moderately overweight BMD of the hip was significantly higher than in those with normal BMI, statistically significant differences of BMC were restricted to females only. Such positive association between body weight and BMC and BMD is in agreement with previous studies on mature subjects, and menopausal and postmenopausal women in particular. In addition, this study demonstrates corresponding positive associations between moderate overweight and bone mass and -density in the elderly and old aged.     

Chemotactic responsiveness toward ligands for CXCR3 and CXCR4 is regulated on plasma blasts during the time course of a memory immune response

Plasma blasts formed during memory immune responses emigrate from the spleen to migrate into the bone marrow and into chronically inflamed tissues where they differentiate into long-lived plasma cells. In this study, we analyze the chemokine responsiveness of plasma blasts formed after secondary immunization with OVA. Starting from day 4 and within approximately 48 h, OVA-specific plasma blasts emigrate from spleen and appear in the bone marrow. Although these migratory cells have lost their responsiveness to many B cell attracting chemokines, e.g., CXC chemokine ligand (CXCL)13 (B lymphocyte chemoattractant), they migrate toward CXCL12 (stromal cell-derived factor 1 alpha), and toward the inflammatory chemokines CXCL9 (monokine induced by IFN-gamma), CXCL10 (IFN-gamma-inducible protein 10), and CXCL11 (IFN-inducible T cell alpha chemoattractant). However, the responsiveness of plasma blasts to these chemokines is restricted to a few days after their emigration from the spleen, indicating a role for these molecules and their cognate receptors, i.e., CXCR3 and CXCR4, in the regulation of plasma blast migration into the bone marrow and/or inflamed tissues.     

Two new loci,PLEIADE and HYADE,implicate organ-specific regulation of cytokinesis in Arabidopsis

In screens for regulators of root morphogenesis in Arabidopsis we isolated six new recessive mutants with irregular cell expansion. Complementation analyses placed the mutations in two loci, PLEIADE (PLE) and HYADE (HYA). Phenotypic analyses revealed multinucleated cells, cell wall stubs, and synchronized cell divisions in incompletely separated cells that are all characteristics of defective cytokinesis. These defects were pronounced in roots and undetectable in aerial organs. In addition, fertility and germination were not affected by the mutations. Thus, the alleles that we have isolated of PLE and HYA suggest that the genes may encode organ-specific components needed primarily during root development. Analysis of microtubule arrays during cell cycle in ple and hya roots indicates that the presence of several synchronized nuclei influences the position of preprophase band, mitotic spindles, and phragmoplasts. The enhanced and synergistic phenotype of PLE/ple.hya/hya seedlings and double mutants point to a role of PLE and HYA in the same process. These mutants provide tools to elucidate the regulation of nuclear cytoskeletal interactions during cell division and cytokinesis.     

Manufacturing DNA microarrays from unpurified PCR products

For the production of DNA microarrays from PCR products, purification of the the DNA fragments prior to spotting is a major expense in cost and time. Also, a considerable amount of material is lost during this process and contamination might occur. Here, a protocol is presented that permits the manufacture of microarrays from unpurified PCR products on aminated surfaces such as glass slides coated with the widely used poly(L-lysine) or aminosilane. The presence of primer molecules in the PCR sample does not increase the non-specific signal upon hybridisation. Overall, signal intensity on arrays made of unpurified PCR products is 94% of the intensity obtained with the respective purified molecules. This slight loss in signal, however, is offset by a reduced variation in the amount of DNA present at the individual spot positions across an array, apart from the considerable savings in time and cost. In addition, a larger number of arrays can be made from one batch of amplification products.     

Molecular identification of a calcium-inhibited catalytic subunit of casein kinase type 2 from Paramecium tetraurelia

We have previously described the occurrence in Paramecium of a casein kinase (CK) activity (EC 2.7.1.37) with some unusual properties, including inhibition by Ca(2+) (R. Kissmehl, T. Treptau, K. Hauser, and H. Plattner, FEBS Lett. 402:227-235, 1995). We now have cloned four genes, PtCK2alpha1 to PtCK2alpha4, all of which encode the catalytic alpha subunit of type 2 CK (CK2) with calculated molecular masses ranging from 38.9 to 39.4 kDa and pI values ranging from 8.8 to 9.0. They can be classified into two groups, which differ from each other by 28% on the nucleotide level and by 18% on the derived amino acid level. One of them, PtCK2alpha3, has been expressed in Escherichia coli and characterized in vitro. As we also have observed with the isolated CK, the recombinant protein preferentially phosphorylates casein but also phosphorylates some Paramecium-specific substrates, including the exocytosis-sensitive phosphoprotein pp63/parafusin. Characteristically, Ca(2+) inhibits the phosphorylation at elevated concentrations occurring during stimulation of a cell. Reconstitution with a recombinant form of the regulatory subunit from Xenopus laevis, XlCK2beta, confirms Ca(2+) sensitivity also under conditions of autophosphorylation. This is unusual for CK2 but correlates with the presence of two EF-hand calcium-binding motifs, one of which is located in the N-terminal segment essential for constitutive activity, as well as with an aberrant composition of normally basic domains recognizing acidic substrate domains. Immunogold localization reveals a considerable enrichment in the outermost cell cortex layers, excluding cilia. We discuss a potential role of this Ca(2+)-inhibited PtCK2alpha species in a late step of signal transduction.     

Augmentation of DNA vaccine potency through secretory heat shock protein-mediated antigen targeting

In an effort to enhance the potency of DNA vaccines, we have developed a new strategy to increase antigen presentation by dendritic cells, one that results in markedly improved cytotoxic T-lymphocyte responses, antibody production, and antitumor effects in vivo. Here, we present the rationale and design of a vaccine encoding a secreted antigen-heat shock protein 70 fusion molecule, targeted to the MHC class I cross-presentation pathway of dendritic cells. Using the human papilloma virus 16 E7 protein as a model antigen, we illustrate the preparation of this vaccine and the main experimental procedures used to test such constructs.     

The tumor necrosis factor-related apoptosis-inducing ligand receptors TRAIL-R1 and TRAIL-R2 have distinct cross-linking requirements for initiation of apoptosis and are non-redundant in JNK activation

Overexpression of the tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) receptors, TRAIL-R1 and TRAIL-R2, induces apoptosis and activation of NF-kappaB in cultured cells. In this study, we have demonstrated differential signaling capacities by both receptors using either epitope-tagged soluble TRAIL (sTRAIL) or sTRAIL that was cross-linked with a monoclonal antibody. Interestingly, sTRAIL was sufficient for induction of apoptosis only in cell lines that were killed by agonistic TRAIL-R1- and TRAIL-R2-specific IgG preparations. Moreover, in these cell lines interleukin-6 secretion and NF-kappaB activation were induced by cross-linked or non-cross-linked anti-TRAIL, as well as by both receptor-specific IgGs. However, cross-linking of sTRAIL was required for induction of apoptosis in cell lines that only responded to the agonistic anti-TRAIL-R2-IgG. Interestingly, activation of c-Jun N-terminal kinase (JNK) was only observed in response to either cross-linked sTRAIL or anti-TRAIL-R2-IgG even in cell lines where both receptors were capable of signaling apoptosis and NF-kappaB activation. Taken together, our data suggest that TRAIL-R1 responds to either cross-linked or non-cross-linked sTRAIL which signals NF-kappaB activation and apoptosis, whereas TRAIL-R2 signals NF-kappaB activation, apoptosis, and JNK activation only in response to cross-linked TRAIL.     

The Finland-United States investigation of non-insulin-dependent diabetes mellitus genetics (FUSION) study. I. An autosomal genome scan for genes that predispose to type 2 diabetes

We performed a genome scan at an average resolution of 8 cM in 719 Finnish sib pairs with type 2 diabetes. Our strongest results are for chromosome 20, where we observe a weighted maximum LOD score (MLS) of 2.15 at map position 69.5 cM from pter and secondary weighted LOD-score peaks of 2.04 at 56.5 cM and 1.99 at 17.5 cM. Our next largest MLS is for chromosome 11 (MLS = 1.75 at 84.0 cM), followed by chromosomes 2 (MLS = 0.87 at 5.5 cM), 10 (MLS = 0.77 at 75.0 cM), and 6 (MLS = 0.61 at 112.5 cM), all under an additive model. When we condition on chromosome 2 at 8.5 cM, the MLS for chromosome 20 increases to 5.50 at 69.0 cM (P=.0014). An ordered-subsets analysis based on families with high or low diabetes-related quantitative traits yielded results that support the possible existence of disease-predisposing genes on chromosomes 6 and 10. Genomewide linkage-disequilibrium analysis using microsatellite marker data revealed strong evidence of association for D22S423 (P=.00007). Further analyses are being carried out to confirm and to refine the location of these putative diabetes-predisposing genes.