One gene but different proteins and diseases: the complexity of dystonin and bullous pemphigoid antigen 1.

Since the immunochemical identification of the bullous pemphigoid antigen 230 (BP230) as one of the major target autoantigens of bullous pemphigoid (BP) in 1981, our understanding of this protein has significantly increased. Cloning of its gene, development and characterization of animal models with engineered gene mutations or spontaneous mouse mutations have revealed an unexpected complexity of the gene encoding BP230. The latter, now called dystonin (DST), is composed of at least 100 exons and gives rise to three major isoforms, an epithelial, a neuronal and a muscular isoform, named BPAG1e (corresponding to the original BP230), BPAG1a and BPAG1b, respectively. The various BPAG1 isoforms play a key role in fundamental processes, such as cell adhesion, cytoskeleton organization, and cell migration. Genetic defects of BPAG1 isoforms are the culprits of epidermolysis bullosa and complex, devastating neurological diseases. In this review, we summarize recent advances of our knowledge about several BPAG1 isoforms, their role in various biological processes and in human diseases.

Shakespeare's Richard II and Henry V and Political Rebellions in the Reign of Queen Elizabeth I

The purpose of this thesis will be to examine how two acts of rebellion against Queen Elizabeth I influenced Shakespeare's writing of Richard II and Henry V, as well as the performance and publication of these plays. The treasonous plots and execution of Mary, Queen of Scots in the 1580s, as well as the failed Essex Rebellion of 1601, resulted in a sensitivity towards any writings that seemed to support a coup d'état. Shakespeare, being a well-informed and fairly well-connected playwright, wrote passages in the afore mentioned plays that clearly reflect the political turmoil of the times. Thus, his plays were censored both on stage and in print until after the death of Elizabeth in 1603.

Mechanism of damage sensing and cell killing following the inhibition of nucleotide excision repair by fludarabine in quiescent cells

Inhibition of DNA repair by the nucleoside of fludarabine (F-ara-A) induces toxicity in quiescent human cells. The sensing and signaling mechanisms following DNA repair inhibition by F-ara-A are unknown. The central hypothesis of this project was that the mechanistic interaction of a DNA repair initiating agent and a nucleoside analog initiates an apoptotic signal in quiescent cells. The purpose of this research was to identify the sensing and signaling mechanism(s) that respond to DNA repair inhibition by F-ara-A. Lymphocytes were treated with F-ara-A, to accumulate the active triphosphate metabolite and subsequently DNA repair was activated by UV irradiation. Pre-incubation of lymphocytes with 3 μM F-ara-A inhibited DNA repair initiated by 2 J/m2 UV and induced greater than additive apoptosis after 24 h. Blocking the incorporation of F-ara-A nucleotide into repairing DNA using 30 μM aphidicolin considerably lowered the apoptotic response. ^ Wild-type quiescent cells showed a significant loss in viability than did cells lacking functional sensor kinase DNA-PKcs or p53 as measured by colony formation assays. The functional status of ATM did not appear to affect the apoptotic outcome. Immunoprecipitation studies showed an interaction between the catalytic sub-unit of DNA-PK and p53 following DNA repair inhibition. Confocal fluorescence microscopy studies have indicated the localization pattern of p53, DNA-PK and γ-H2AX in the nucleus following DNA damage. Foci formation by γ-H2AX was seen as an early event that is followed by interaction with DNA-PKcs. p53 serine-15 phosphorylation and accumulation were detected 2 h after treatment. Fas/Fas ligand expression increased significantly after repair inhibition and was dependent on the functional status of p53. Blocking the interaction between Fas and Fas ligand by neutralizing antibodies significantly rescued the apoptotic fraction of cells. ^ Collectively, these results suggest that incorporation of the nucleoside analog into repair patches is critical for cytotoxicity and that the DNA damage, while being sensed by DNA-PK, may induce apoptosis by a p53-mediated signaling mechanism. Based on the results, a model is proposed for the sensing of F-ara-A-induced DNA damage that includes γ-H2AX, DNA-PKcs, and p53. Targeting the cellular DNA repair mechanism can be a potential means of producing cytotoxicity in a quiescent population of neoplastic cells. These results also provide mechanistic support for the success of nucleoside analogs with cyclophosphamide or other agents that initiate excision repair processes, in the clinic. ^

Notes to Volumes I and II : from the creation to the exodus

by Louis Ginzberg

Physical activity, body composition, exogenous estrogen use and their association with insulin-like growth factors

Obesity and physical inactivity are modifiable risk factors that are associated with several health issues; they are major factors in up to 30% of major cancers. Elevated levels of circulating insulin-like growth factor-I (IGF-I) have been associated with high body composition measurements and high cancer risk; exogenous estrogen use is associated with low circulating IGF-I levels and high cancer risk. The relationship between physical activity and circulating IGF levels is complex and findings of previous studies of their relationship remain inconsistent; however, these studies included vague definitions of physical activity. In this study, we used cross-sectional data from the Women's Health Initiative to determine the relationship between specific measures of physical activity (e.g., intensity, duration, and frequency) and circulating IGF-I levels, accounting for exogenous estrogen use and body composition. These data were collected from women enrolled at Women's Health Initiative clinical centers at Baylor College of Medicine and Wake Forest University School of Medicine. Multivariate linear regression analysis showed that circulating IGF-I and IGF-binding protein (BP) 3 levels were positively associated with frequency, duration, and intensity of physical activity. Circulating IGF-I levels and the molar IGF-I:IGF-BP3 ratio were significantly associated with frequency of walking, whereas circulating IGF-BP3 levels were significantly associated with strenuous physical activity, suggesting that different aspects of physical activity and their effects on fitness affect members of the IGF family differently. The results from our study support the recommendation of a regular exercise routine, particularly that of strenuous intensity, for postmenopausal women as a means to prevention of cancer.^

BIOLOGICAL MECHANISMS AND CLINICAL IMPLICATIONS OF BCR-ABL-INDUCED MITOCHONDRIAL OXIDATIVE STRESS AND CELL SURVIVAL IN CHRONIC MYELOID LEUKEMIA

Chronic myeloid leukemia (CML), a myeloproliferative disorder, represents approximately 15-20% of all adult leukemia. The development of CML is clearly linked to the constitutively active protein-tyrosine kinase BCR-ABL, which is encoded by BCR-ABL fusion gene as the result of chromosome 9/22 translocation (Philadelphia chromosome). Previous studies have demonstrated that oxidative stress-associated genetic, metabolic and biological alterations contribute to CML cell survival and drug refractory. Mitochondria and NAD(P)H oxidase (NOX) are the major sources of BCR-ABL-induced cellular reactive oxygen species (ROS) production. However, it is still unknown how CML cells maintain the altered redox status, while escaping from the persistent oxidative stress-induced cell death. Therefore, elucidation of the mechanisms by which CML cells cope with oxidative stress will provide new insights into CML leukemogenesis. The major goal of this study is to identify the survival factors protecting CML cells against oxidative stress and develop novel therapeutic strategies to overcome drug resistance. Several experimental models were used to test CML cell redox status and cellular sensitivity to oxidative stress, including BCR-ABL inducible cell lines, BCR-ABL stably transformed cell lines and BCR-ABL-expressing CML blast crisis cells with differential BCL-XL/BCL-2 expressions. Additionally, an artificial CML cell model with heterogenic BCL-XL/BCL-2 expression was established to assess the correlation between differential survival factor expression patterns and cell sensitivity to Imatinib and oxidative stress. In this study, BCL-XL and GSH have been identified as the major survival factors responsive to BCR-ABL-promoted cellular oxidative stress and play a dominant role in regulating the threshold of oxidative stress-induced apoptosis. Cell survival factors BCL-XL and BCL-2 differentially protect mitochondria under oxidative stress. BCL-XL is an essential survival factor in preventing excessive ROS-induced cell death while BCL-2 seems to play a relatively minor role. Furthermore, the redox modulating reagent β-phenethyl isothiocyanate (PEITC) has been found to efficiently deplete GSH and induce potent cell killing effects in drug-resistant CML cells. Combination of PEITC with BCL-XL/BCL2 inhibitor ABT737 or suppression of BCL-XL by BCR-ABL inhibitor Gleevec dramatically sensitizes CML cells to apoptosis. These results have suggested that elevation of BCL-XL and cellular GSH are important for the development of CML, and that redox-directed therapy is worthy of further clinical investigations in CML.

NMR structural analysis of cardiac troponin C: Monitoring conformational changes induced by binding calcium, troponin I, and calcium -sensitizing drugs

Contraction of cardiac muscle is regulated through the Ca2+ dependent protein-protein interactions of the troponin complex (Tn). The critical role cardiac troponin C (cTnC) plays as the Ca2+ receptor in this complex makes it an attractive target for positive inotropic compounds. In this study, the ten Met methyl groups in cTnC, [98% 13C ϵ]-Met cTnC, are used as structural markers to monitor conformational changes in cTnC and identify sites of interaction between cTnC and cardiac troponin I (cTnI) responsible for the Ca2+ dependent interactions. In addition the structural consequences that a number of Ca2+-sensitizing compounds have on free cTnC and the cTnC·cTnI complex were characterized. Using heteronuclear NMR experiments and monitoring chemical shift changes in the ten Met methyl 1H-13C correlations in 3Ca2+ cTnC when bound to cTnI revealed an anti-parallel arrangement for the two proteins such that the N-domain of cTnI interacts with the C-domain of cTnC. The large chemical shifts in Mets-81, -120, and -157 identified points of contact between the proteins that include the C-domain hydrophobic surface in cTnC and the A, B, and D helical interface located in the regulatory N-domain of cTnC. TnI association [cTnI(33–80), cTnI(86–211), or cTnI(33–211)] was found also to dramatically reduce flexibility in the D/E central linker of cTnC as monitored by line broadening in the Met 1H- 13C correlations of cTnC induced by a nitroxide spin label, MTSSL, covalently attached to cTnC at Cys 84. TnI association resulted in an extended cTnC that is unlike the compact structure observed for free cTnC. The Met 1H-13C correlations also allowed the binding characteristics of bepridil, TFP, levosimendan, and EMD 57033 to the apo, 2Ca2+, and Ca2+ saturated forms of cTnC to be determined. In addition, the location of drug binding on the 3Ca2+cTnC·cTnI complex was identified for bepridil and TFP. Use of a novel spin-labeled phenothiazine, and detection of isotope filtered NOEs, allowed identification of drug binding sites in the shallow hydrophobic cup in the C-terminal domain, and on two hydrophobic surfaces on N-regulatory domain in free 3Ca2+ cTnC. In contrast, only one N-domain drug binding site exists in 3Ca2+ cTnC·cTnI complex. The methyl groups of Met 45, 60 and 80, which are grouped in a hydrophobic patch near site II in cTnC, showed the greatest change upon titration with bepridil or TFP, suggesting that this is a critical site of drug binding in both free cTnC and when associated with cTnI. The strongest NOEs were seen for Met-60 and -80, which are located on helices C and D, respectively, of Ca2+ binding site II. These results support the conclusion that the small hydrophobic patch which includes Met-45, -60, and -80 constitutes a drug binding site, and that binding drugs to this site will lead to an increase in Ca2+ binding affinity of site II while preserving maximal cTnC activity. Thus, the subregion in cTnC makes a likely target against which to design new and selective Ca2+-sensitizing compounds. ^

Curie temperatures and cell edges of ODP Leg 120 samples (Table 1)

Basalt samples recovered on Ocean Drilling Program Leg 120 from the Kerguelen Plateau were investigated by thermomagnetic analysis, X-ray diffraction, and ore microscopy. The basement samples could be divided into two groups based on Curie temperatures, cell-edge parameters, and optical magnetic mineralogy. Samples from Sites 748 and 750 underwent only low-temperature oxidation and displayed Curie temperatures for the titanomaghemites that ranged from 340° to 395°C. The basalts from Sites 747 and 749 mainly experienced high-temperature oxidation. High-temperature oxidation produced titanium-poor titanomagnetites with ilmenite-exsolution lamellae. Curie temperatures of the deuterically oxidized titanomaghemites varied from 490° to 620°C.

Water temperature reconstruction for Terminations I and II of sediment core MD01-2378

We present centennial records of sea surface and upper thermocline temperatures in Core MD01-2378 from the Timor Sea, which provide new insights into the variability of the Indonesian outflow across the last two glacial terminations. Mg/Ca in Globigerinoides ruber (white s. s.) indicates an overall increase of 3.2 °C in sea surface temperature (SST) over Termination I. Following an early Holocene plateau at 11.3-6.4 ka, SSTs cooled by 0.6 °C during the middle to late Holocene (6.4-0.7 ka). The early Holocene warming occurred in phase with increasing northern hemisphere summer insolation, coinciding with northward displacement of the Intertropical Convergence Zone, enhanced boreal summer monsoon and expansion of the Indo-Pacific Warm Pool. Thermocline temperatures (Pulleniatina obliquiloculata Mg/Ca) gradually decreased from 24.5 to 21.5 °C since 10.3 ka, reflecting intensification of a cool thermocline throughflow. The vertical structure of the upper ocean in the Timor Sea evolved in similar fashion during the Holocene and MIS5e, although the duration of SST plateaux differed (11.3 to 6.4 ka in Termination I and from 129 to 119 ka in Termination II), which was probably due to the more intense northern hemisphere summer insolation during MIS 5e. During both terminations, SST increased simultaneously in the southern high latitudes and the tropical eastern Indian Ocean, suggesting virtually instantaneous atmospheric climate feedbacks between the high and low latitudes.