Disease-associated pathophysiologic structures in pediatric rheumatic diseases show characteristics of scale-free networks seen in physiologic systems: implications for pathogenesis and treatment

Background

Tamoxifen (TAM) is a well characterized breast cancer drug and selective estrogen receptor modulator (SERM) which also has been associated with a small increase in risk for uterine cancers. TAM's partial agonist activation of estrogen receptor has been characterized for specific gene promoters but not at the genomic level in vivo.Furthermore, reducing uncertainties associated with cross-species extrapolations of pharmaco- and toxicogenomic data remains a formidable challenge.

Results

A comparative ligand and species analysis approach was conducted to systematically assess the physiological, morphological and uterine gene expression alterations elicited across time by TAM and ethynylestradiol (EE) in immature ovariectomized Sprague-Dawley rats and C57BL/6 mice. Differential gene expression was evaluated using custom cDNA microarrays, and the data was compared to identify conserved and divergent responses. 902 genes were differentially regulated in all four studies, 398 of which exhibit identical temporal expression patterns.

Conclusion

Comparative analysis of EE and TAM differentially expressed gene lists suggest TAM regulates no unique uterine genes that are conserved in the rat and mouse. This demonstrates that the partial agonist activities of TAM extend to molecular targets in regulating only a subset of EE-responsive genes. Ligand-conserved, species-divergent expression of carbonic anhydrase 2 was observed in the microarray data and confirmed by real time PCR. The identification of comparable temporal phenotypic responses linked to related gene expression profiles demonstrates that systematic comparative genomic assessments can elucidate important conserved and divergent mechanisms in rodent estrogen signalling during uterine proliferation.     

High-energy phosphates and tension production in rabbit tibialis anterior/extensor digitorum longus muscles

Ryschon, T. W., J. C. Jarvis, S. Salmons, and R. S. Balaban.High-energy phosphates and tension production in rabbit tibialisanterior/extensor digitorum longus muscles. J. Appl. Physiol. 82(3): 1024-1029, 1997.The effects ofrepetitive muscle contraction on energy state and tension productionwere studied in rabbit tibialis anterior/extensor digitorum longusmuscles that had been subjected to 90 days of continuous indirectelectrical stimulation at 10 Hz. Anesthetized chronically stimulatedand control rabbits were challenged with 15 min of stimulation at 4 and15 tetani/min.P_i-to-phosphocreatine (PCr) ratio(P_i/PCr) was measured in vivo before, during, andafter acute stimulation by³¹P-magnetic resonancespectroscopy, and tension was recorded at the same time. AlthoughP_i/PCr was low at rest, it wassignificantly higher in chronically stimulated muscle than in controlmuscle (0.20 ± 0.02 vs. 0.05 ± 0.01, P < 0.05). Stimulation of control muscle for 15 min at both 4 and 15 tetani/min induced a significant rise in P_i/PCr, whereas the sameconditions in chronically stimulated muscle did not produce anysignificant departure from initial levels. The tension produced bycontrol muscle fell to 93 ± 3% of its initial value duringstimulation at 4 tetani/min and to 61 ± 7% at 15 tetani/min,respectively. In chronically stimulated muscle, on the other hand,tension was potentiated above its initial level at both stimulationrates (135 ± 15 and 138 ± 11%, respectively) and remainedsignificantly elevated throughout each trial. The ability ofchronically stimulated muscle to sustain high levels of activity withminimal perturbations in P_i/PCr ordecrement in tension is attributable to cellular adaptations thatinclude a well-documented increase in oxidative capacity.

     

Light-Sensitivity of Hazel Seeds with Respect to the Breaking of Dormancy

Light breaks the dormancy of hazel (Corylus avellana L.) seedswhen the testa is removed. Light, acting via phytochrome, alsopromotes the germination of hazel seeds when the embryonic axisis surgically uncovered. In such seeds with the axis uncoveredremoval of the testa is antagonistic to the light promotionof germination. (Received January 17, 1982; Accepted April 25, 1983)     

Baculovirus phosphoprotein pp31 is associated with virogenic stroma.

The PstI K fragment of Autographa californica nuclear polyhedrosis virus (AcMNPV) encodes a protein with a molecular weight of 31,000. To define the role of this protein (pp31) in virus infection further, it was overexpressed in bacteria and used to produce polyclonal antiserum. Radioimmunoprecipitation analysis indicated that pp31 was synthesized during both the early and late phases of virus infection, consistent with previous analyses indicating that the gene was regulated by tandem early and late promoters. Metabolic labeling of cells with carrier-free phosphate indicated that pp31 was phosphorylated. Biochemical fractionation experiments showed that pp31 was localized in the nucleus and that it was more stably associated with the nucleus at later times of infection. Immunoblot analysis of subnuclear fractions indicated that pp31 was associated predominantly with the chromatin and nuclear matrix fractions. Immunofluorescence experiments confirmed that the pp31 protein was localized in the nucleus. Nuclear staining was relatively uniform early but was more centrally nuclear later in infection. Immunoelectron microscopy indicated that the pp31 protein was a component of virogenic stroma. Southwestern (DNA-protein) blot analysis demonstrated that pp31 is a DNA-binding protein. These findings suggest a possible role for pp31 in the virus life cycle.     

Structural basis for the platelet-collagen interaction: the smallest motif within collagen that recognizes and activates platelet Glycoprotein VI contains two glycine-proline-hydroxyproline triplets

Collagen-related peptide is a selective agonist for the platelet collagen receptor Glycoprotein VI. The triple helical peptide contains ten GPO triplets/strand (single letter amino acid nomenclature, where O is hydroxyproline) and so over-represents GPO compared with native collagen sequence. To investigate the ability of Glycoprotein VI to recognize GPO triplets in a setting more representative of the collagens, we synthesized a set of triple helical peptides containing fewer GPO triplets, varying their number and spacing within an inert (GPP)n backbone. The adhesion of recombinant human Glycoprotein VI ectodo-main, like that of human platelets, to these peptides increased with their GPO content, and platelet adhesion was abolished by the specific anti-Glycoprotein VI-blocking antibody, 10B12. Platelet aggregation and protein tyrosine phosphorylation were induced only by cross-linked peptides and only those that contained two or more GPO triplets. Such peptides were less potent than cross-linked collagen-related peptide. Our data suggest that both the sequences GPOGPO and GPO.........GPO represent functional Glycoprotein VI recognition motifs within collagen. Furthermore, we propose that the (GPO)4 motif can support simultaneous binding of two glycoprotein VI molecules, in either a parallel or anti-parallel stacking arrangement, which could play an important role in activation of signaling.     

Kaposi's sarcoma-associated herpesvirus-induced upregulation of the c-kit proto-oncogene,as identified by gene expression profiling,is essential for the transformation of endothelial cells

Kaposi's sarcoma (KS), the most frequent malignancy afflicting AIDS patients, is characterized by spindle cell formation and vascularization. Infection with KS-associated herpesvirus (KSHV) is consistently observed in all forms of KS. Spindle cell formation can be replicated in vitro by infection of dermal microvascular endothelial cells (DMVEC) with KSHV. To study the molecular mechanism of this transformation, we compared RNA expression profiles of KSHV-infected and mock-infected DMVEC. Induction of several proto-oncogenes was observed, particularly the receptor tyrosine kinase c-kit. Consistent with increased c-Kit expression, KHSV-infected DMVEC displayed enhanced proliferation in response to the c-Kit ligand, stem cell factor (SCF). Inhibition of c-Kit activity with either a pharmacological inhibitor of c-Kit (STI 571) or a dominant-negative c-Kit protein reversed SCF-dependent proliferation. Importantly, inhibition of c-Kit signal transduction reversed the KSHV-induced morphological transformation of DMVEC. Furthermore, overexpression studies showed that c-Kit was sufficient to induce spindle cell formation. Together, these data demonstrate an essential role for c-Kit in KS tumorigenesis and reveal a target for pharmacological intervention.     

Strontium-89 and Strontium-90 Levels in Breast Milk and in Mineral-Supplement Preparations

Strontium-90, strontium-89 and S.U. values were determined in human milk before and after the resumption of atmospheric nuclear testings in 1961, and the levels were compared to cows'' milk values reported during the same time. S.U.⁹⁰ levels in human milk were approximately one-fifth of those found in cows'' milk. Assuming an average dietary intake of 11-13 S.U.⁹⁰ during the period tested, the mean strontium/calcium ratio of 1.78 found in human milk represents an Observed Ratio milk-diet of approximately 0.14-0.16. Although strontium-89 was present in cows'' milk already in September 1961, it did not appear in human milk until November 1961. It seems, therefore, that there was a two-month lag period between the appearance of fresh fallout in cows'' milk and human milk. Calcium-supplement mineral preparations used by pregnant and lactating women were tested to find their strontium-89, strontium-90 and S.U. levels, because strontium isotopes, if present in these products, will be transferred to the fetus and to breast-fed infants. The compounds tested had S.U.⁹⁰ levels of 0.13-2.62; in none of the preparations was Sr⁸⁹ present.     

A novel yeast expression system for the overproduction of quality-controlled membrane proteins

Saturation of the cell's protein folding capacity and accumulation of inactive incompletely folded protein often accompanying the overexpression of membrane proteins (MPs) presents an obstacle to their efficient purification in a functional form for structural studies. We present a novel strategy for optimization of functional MP expression in Saccharomyces cerevisiae. This approach exploits the unfolded protein response (UPR) pathway, a stress signaling mechanism that senses the accumulation of unfolded proteins in the endoplasmic reticulum. We demonstrate that a high level of UPR induction upon expression of a MP reflects impaired functional expression of that protein. Tuning the expression level of the protein so as to avoid or minimize UPR induction results in its increased functional expression. UPR status can therefore serve as a proxy variable for the extent of impaired expression of a MP that may even be applicable in the absence of knowledge of the protein's biological function.     

Pre-natal stress amplifies the immediate behavioural responses to acute pain in piglets

Pre-natal stress (PNS) or undernutrition can have numerous effects on an individual''s biology throughout their lifetime. Some of these effects may be adaptive by allowing individuals to tailor their phenotype to environmental conditions. Here we investigated, in the domestic pig Sus scrofa, whether one possible consequence of a predicted adverse environment could be altered pain perception. The behavioural response of piglets to the surgical amputation (‘docking’) of their tail or a sham procedure was measured for 1 min in piglets born to mothers who either experienced mid-gestation social stress or were left undisturbed throughout pregnancy. A behavioural pain score was found to predict the docked status of piglets with high discriminant accuracy. Piglets exposed to PNS had a significantly higher pain score than controls, and for each litter of tail-docked piglets, the average pain score was correlated with mid-gestation maternal cortisol levels. The data presented here provide evidence that the experience of stress in utero can result in a heightened acute response to injury in early life. Speculatively, this may represent an adaptive alteration occurring as a consequence of a pre-natal ‘early warning’ of environmental adversity.