Calcium and collagen binding properties of osteopontin, bone sialoprotein, and bone acidic glycoprotein-75 from bone.

Calcium binding properties of bone acidic glycoprotein-75, osteopontin, and bone sialoprotein were determined in 10 mM imidazole buffer (pH 6.8), containing either 60 mM KCl or 150 mM NaCl. Proteins assayed were first bound to nitrocellulose to mimic substrate-bound forms in vivo; retention of phosphoproteins was determined through use of radioiodinated tracers. Binding studies were carried out both as a function of calcium concentration and the amount of phosphoprotein. In the presence of 60 mM KCl, bone acidic glycoprotein-75 exhibited the largest calcium binding capacity (139 atoms/molecule at saturation), with bone sialoprotein intermediary (83 atoms/molecule) and osteopontin lowest (50 atoms/molecule). Sites detected for each phosphoprotein exhibited overall binding constants in the 0.5-1.0 mM extracellular range. In 150 mM NaCl and 1-2 mM total calcium, phosphoproteins bound between 72 and 19 mol of calcium/mol with the same relative order. Binding was proportional to amount of phosphoprotein in either salt condition. The presence of 5 mM calcium had a different effect on concentration-dependent binding to type I collagen for each phosphoprotein. Bone acidic glycoprotein-75 alone was found to undergo an unusual calcium-enhanced polymerization reaction, confirmed by light scattering measurements, wherein collagen binding was greatest with polymeric forms. These findings demonstrate that acidic phosphoproteins from bone bind calcium atoms with a range of capacities. Calcium appears to induce conformational changes in bone acidic glycoprotein-75 which influences its self-association and binding to different substrata.     

Solid cancer incidence among the Chernobyl emergency workers residing in Russia: estimation of radiation risks

An analysis is presented of solid cancer incidence during 11 years of follow-up (1991–2001) of Chernobyl emergency workers residing in Russia. The analysis is based on data from the cohort of male emergency workers from 6 regions in Russia including 55,718 persons with documented external radiation doses in the range of 0.001–0.3 Gy who worked within the 30 -km zone in 1986–1987. The mean age at exposure for these persons was 34.8 years and the mean external radiation dose 0.13 Gy. In the cohort 1,370 cases of solid cancer were diagnosed and 3 follow-up periods were considered: 1991–1995, 1996–2001 and 1991–2001. The second follow-up period was chosen to allow for a minimum latency period of 10 years being characteristic of solid cancers. For risk assessment two control groups have been introduced, the first external one representing incidence rates for corresponding ages in Russia in general, the second internal one consisting of emergency workers. The risk estimates were based on spontaneous incidence rates of solid cancer. The estimated standardized incidence ratio (SIR) is in good agreement (95% CI) with that of the control. The values of excess relative risk per unit dose (ERR/Gy) for solid malignant neoplasms have been estimated to be 0.33 (95% CI: –0.39, 1.22) (internal control) for the follow-up period 1991–2001 and 0.19 (95% CI: –0.66, 1.27) for 1996–2001.     

Global expression profiling of theophylline response genes in macrophages: evidence of airway anti-inflammatory regulation

Background

Theophylline has been used widely as a bronchodilator for the treatment of bronchial asthma and has been suggested to modulate immune response. While the importance of macrophages in asthma has been reappraised and emphasized, their significance has not been well investigated. We conducted a genome-wide profiling of the gene expressions of macrophages in response to theophylline.

Methods

Microarray technology was used to profile the gene expression patterns of macrophages modulated by theophylline. Northern blot and real-time quantitative RT-PCR were also used to validate the microarray data, while Western blot and ELISA were used to measure the levels of IL-13 and LTC4.

Results

We identified dozens of genes in macrophages that were dose-dependently down- or up-regulated by theophylline. These included genes related to inflammation, cytokines, signaling transduction, cell adhesion and motility, cell cycle regulators, and metabolism. We observed that IL-13, a central mediator of airway inflammation, was dramatically suppressed by theophylline. Real-time quantitative RT-PCR and ELISA analyses also confirmed these results, without respect to PMA-treated THP-1 cells or isolated human alveolar macrophages. Theophylline, rolipram, etazolate, db-cAMP and forskolin suppressed both IL-13 mRNA expression (~25%, 2.73%, 8.12%, 5.28%, and 18.41%, respectively) and protein secretion (<10% production) in macrophages. These agents also effectively suppressed LTC4 expression.

Conclusion

Our results suggest that the suppression of IL-13 by theophylline may be through cAMP mediation and may decrease LTC4 production. This study supports the role of theophylline as a signal regulator of inflammation, and that down regulation of IL-13 by theophylline may have beneficial effects in inflammatory airway diseases.     

Differential effects of the perinatal steroid environment on three sexually dimorphic parameters of the rat brain

Gonadectomy of male rats was performed at 0, 6-7 (6h), 12-13 (12h), or 24 h postnatally in order to examine the influence of testosterone exposure on sexual differentiation of the brain. The indices examined were: the volume of the sexually dimorphic nucleus of the preoptic area (SDN-POA) and luteinizing hormone (LH) and follicle-stimulating hormone (FSH) titers following estradiol benzoate (EB) and progesterone (P) administration. Control animals were sham-operated at 0 h and gonadectomized at 29 days of age (sham). A decrease in the percentage of males with elevated plasma LH levels following P was found with increasing delay before gonadectomy. Significant (P less than 0.001) differences existed in the amplitude of plasma LH titers 5 h following P administration between sham, 0 h, and 6 h groups. Follicle-stimulating hormone was also elevated in all neonatally gonadectomized male groups following P administration, but there was no difference between the groups. Volume of the SDN-POA was significantly (P less than 0.001) smaller in all gonadectomized males when compared to that of sham-operated males, but no differences existed between males gonadectomized at the different hours postpartum. In female rats gonadectomized at 0 h (F0h), LH levels were elevated 5 h following P, but only to a magnitude of 36% of that of sham-operated controls (P less than 0.001). Volume of the SDN-POA of the F0h group was significantly reduced (P less than 0.05) when compared to that of sham females. Thus, in males, the presence of the tests prenatally may be responsible for the initiation of masculinization of LH release mechanisms and the SDN-POA, but both require further androgen exposure for their completion. In addition, the LH and FSH regulating systems show a differential sensitivity to the steroid hormone environment during development that shapes the animal's response to steroid as an adult.     

Marked effects of salt on estrogen receptor binding to DNA: biologically relevant discrimination between DNA sequences

Avidin-biotin complexed with DNA (ABCD) assays were employed to determine the binding affinity of estrogen receptor (ER) to DNA under various salt conditions. Type and concentration of salt in the reaction buffer dramatically affected the ability of the ER to discriminate between DNA sequences. Under appropriate salt conditions, ER was able to bind to the estrogen response element from the Xenopus vitellogenin A2 gene with at least 3 orders of magnitude greater affinity than a two base pair mutant sequence, and 5 orders of magnitude greater affinity than plasmid DNA. In these studies, the best discrimination was observed under conditions of salt type and concentration that more closely approximated intracellular conditions, i.e., 100-150 mM potassium salts. Analysis of the binding affinities for ER to all three types of DNA over a range of KCl concentrations indicated that the ionic interactions upon ER binding were the same for the three DNA molecules tested. Therefore, the additional stability of ER binding to target DNA sequences was contributed by nonionic interactions.     

Complex T cell memory repertoires participate in recall responses at extremes of antigenic load

The CD8 T cell memory response to the HLA-A2-restricted influenza epitope M1(58-66) can be an instructive model of immune memory to a nonevolving epitope of a frequently encountered pathogen that undergoes clearance. This memory repertoire can be complex, composed of a large number of clonotypes represented at low copy numbers, while maintaining a focus on the use of VB17 T cell receptors with identified Ag recognition motifs. Such a repertoire structure might provide a panoply of clonotypes whose differential avidity for the epitope would allow responses under varying antigenic loads. This possibility was tested experimentally by characterizing the responding repertoire in vitro while varying influenza Ag concentration over five orders of magnitude. At higher and lower Ag concentrations there was increased cell death, yet a focused but diverse response could still be observed. Thus, one of the characteristics of complex memory repertoires is to provide effector function at extremes of Ag load, a characteristic that is not generally considered in vaccination development but may be important in measuring its efficacy.     

Cooperativity of hydrophobic anchor interactions: evidence for epitope selection by MHC class II as a folding process

Peptide binding to MHC class II (MHCII) molecules is stabilized by hydrophobic anchoring and hydrogen bond formation. We view peptide binding as a process in which the peptide folds into the binding groove and to some extent the groove folds around the peptide. Our previous observation of cooperativity when analyzing binding properties of peptides modified at side chains with medium to high solvent accessibility is compatible with such a view. However, a large component of peptide binding is mediated by residues with strong hydrophobic interactions that bind to their respective pockets. If these reflect initial nucleation events they may be upstream of the folding process and not show cooperativity. To test whether the folding hypothesis extends to these anchor interactions, we measured dissociation and affinity to HLA-DR1 of an influenza hemagglutinin-derived peptide with multiple substitutions at major anchor residues. Our results show both negative and positive cooperative effects between hydrophobic pocket interactions. Cooperativity was also observed between hydrophobic pockets and positions with intermediate solvent accessibility, indicating that hydrophobic interactions participate in the overall folding process. These findings point out that predicting the binding potential of epitopes cannot assume additive and independent contributions of the interactions between major MHCII pockets and corresponding peptide side chains.