Solution structure(s) of trinucleosomes from contrast variation SAXS

Nucleosomes in all eukaryotic cells are organized into higher order structures that facilitate genome compaction. Visualizing these organized structures is an important step in understanding how genomic DNA is efficiently stored yet remains accessible to information-processing machinery. Arrays of linked nucleosomes serve as useful models for understanding how the properties of both DNA and protein partners affect their arrangement. A number of important questions are also associated with understanding how the spacings between nucleosomes are affected by the histone proteins, chromatin remodelers, or other chromatin-associated protein partners. Contrast variation small angle X-ray scattering (CVSAXS) reports the DNA conformation within protein-DNA complexes and here is applied to measure the conformation(s) of trinucleosomes in solution, with specific sensitivity to the distance between and relative orientation of linked nucleosomes. These data are interpreted in conjunction with DNA models that account for its sequence dependent mechanical properties, and Monte-Carlo techniques that generate realistic structures for comparison with measured scattering profiles. In solution, trinucleosomes segregate into two dominant populations, with the flanking nucleosomes stacked or nearly equilaterally separated, e.g. with roughly equal distance between all pairs of nucleosomes. These populations are consistent with previously observed magnesium-dependent structures of trinucleosomes with shorter linkers.     

Epidermal growth factor plasma concentrations in healthy control persons, acute and chronic stress and during pregnancy.

Plasma concentrations of Epidermal Growth Factor (EGF) in healthy control persons do not change significantly during adult lifetime and no diurnal changes could be detected. In addition, no alterations of plasma EGF concentrations occur during acute and chronic stress and in all three trimesters of pregnancy. Thus, the previously suggested theories that EGF might play a causative role in the activation of the hypothalamic-pituitary adrenal axis during the stress reaction and in the prevention of peptic ulcers in pregnancy could not be confirmed by the present data.     

Autocrine growth limitation of human papillomavirus type 16-harboring keratinocytes by constitutively released tumor necrosis factor-alpha.

TNF-alpha is known to exert antitumor and antiviral effects and to participate in the regulation of the immune response. In our study we demonstrate that human rTNF-alpha specifically blocks growth of SK-v keratinocyte cell line harboring and expressing human papillomavirus type 16 (HPV16) sequences. This inhibitory effect was shown by [3H]TdR incorporation and cell counting. Binding experiments with 125I-TNF-alpha showed that SK-v cells express about 10,000 single class TNF-alpha R per cell with affinity constant of about 0.7 nM. Binding of 125I-TNF-alpha could be inhibited by htr-9 mAb recognizing a 55/60-kDa type I TNF-alpha R but not by utr-1 mAb recognizing 75/80-kDa type II TNF-alpha R or irrelevant mAb specific for HPV16E7 protein. Addition of anti-TNF-alpha antibodies to SK-v cell culture resulted in significant (p < 0.05), dose-dependent stimulation of their proliferation. SK-v cells constitutively expressed TNF-alpha mRNA, and SK-v CM contained TNF-alpha, as demonstrated by Northern blot analysis, a specific ELISA, Western blot analysis, and a bioassay with TNF-alpha-sensitive L-M cells. HPLC gel filtration of SK-v cell CM showed that the factor cytotoxic for L-M cells coeluted with immunoreactive TNF-alpha. These results demonstrate that HPV16-harboring SK-v cells constitutively express and release immunoreactive and biologically active TNF-alpha that in turn may exert an autocrine growth inhibitory effect. This phenomenon could represent one of the self-limiting mechanisms controling growth of HPV-induced neoplasia.     

Interleukin-1 protects transformed keratinocytes from tumor necrosis factor-related apoptosis-inducing ligand- and CD95-induced apoptosis but not from ultraviolet radiation-induced apoptosis.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a new member of the tumor necrosis factor (TNF) family, induces apoptosis primarily of transformed cells. Interleukin-1 was previously found to protect the keratinocyte cell line KB from TRAIL-induced apoptosis, thus we studied whether interleukin-1 also protects from other apoptotic stimuli (ultraviolet radiation (UV), CD95-ligand). Interleukin-1 rescued KB cells from TRAIL- and CD95-induced apoptosis, which was critically dependent on nuclear factor kappaB, because cells transfected with a super-repressor form of the nuclear factor kappaB inhibitor IkappaB were less protected. In contrast, UV-mediated apoptosis was not only not prevented by interleukin-1 but even enhanced. This opposite effect of interleukin-1 was also observed for the expression of the inhibitor of apoptosis proteins (IAP). Whereas TRAIL- and CD95-mediated suppression of IAP expression was partially reversed by interleukin-1, UV-mediated down-regulation of IAPs was not reversed but even further enhanced. Increased apoptosis induced by interleukin-1 plus UV was accompanied by excessive TNFalpha release, implying that enhanced cytotoxicity is due to the additive effect of these two apoptotic stimuli. Accordingly, enhanced apoptosis was reduced by blocking the TNF receptor-1. The opposite effects of interleukin-1 indicate that different mechanisms are involved in UV-induced apoptosis compared with CD95- and TRAIL-mediated apoptosis. Furthermore, the data suggest that whether a signal acts in an antiapoptotic way or not does not only depend on the signal itself but also on the stimulus causing apoptosis.     

Acute exercise stimulates the renin-angiotensin-aldosterone axis: adaptive changes in runners

The plasma concentrations of aldosterone and its known regulators, plasma renin, potassium and ACTH, were examined during graded intensities of treadmill exercise (50, 70 and 90% of maximal oxygen uptake, VO2max). Sedentary men (n = 7) and two groups of runners of different training status (moderately trained, 15-25 miles/week, n = 7; highly trained, greater than 45 miles/week, n = 7) were studied in an attempt to define whether physical training causes changes in aldosterone homeostasis. Acute exercise was associated with elevations in plasma aldosterone, renin activity, potassium and ACTH in all three groups of subjects at exercise intensities of 70 and 90% VO2max. There were no differences in any of the responses among the three groups except for a blunted response of PRA at 90% VO2max in highly trained athletes. The exercise-induced rise of plasma aldosterone concentration did not correlate with changes in the concentration of its regulatory substances. We conclude that exercise stimulates the renin-angiotensin-aldosterone axis in an intensity-dependent fashion. With increased physical training identical hormonal and metabolic responses result at increased absolute workloads.