Structural elucidation and antisense properties of a sugar-modified DNA analogue

Antisense oligonucleotides deserve great attention as potential drug candidates for the treatment of genetic disorders. For example, muscle dystrophy can be treated successfully in mice by antisense-induced exon skipping in the pre-mRNA coding for the structural protein dystrophin in muscle cells. For this purpose a sugar- and backbone-modified DNA analogue was designed, in which a tricyclic ring system substitutes the deoxyribose. These chemical modifications stabilize the dimers formed with the targeted RNA relative to native nucleic acid duplexes and increase the biostability of the antisense oligonucleotide. While evading enzymatic degradation constitutes an essential property of antisense oligonucleotides for therapeutic application, it renders the oligonucleotide inaccessible to biochemical sequencing techniques and requires the development of alternative methods based on mass spectrometry. The set of sequences studied includes tcDNA oligonucleotides ranging from 10 to 15 nucleotides in length as well as their hybrid duplexes with DNA and RNA complements. All samples were analyzed on a LTQ Orbitrap XL instrument equipped with a nano-electrospray source. For tandem mass spectrometric experiments collision-induced dissociation was performed, using helium as collision gas. Mass spectrometric sequencing of tcDNA oligomers manifests the applicability of the technique to substrates beyond the scope of enzyme-based methods. Sequencing requires the formation of characteristic backbone fragments, which take the form of a-B- and w-ions in the product ion spectra of tcDNA. These types of product ions are typically associated with unmodified DNA, which suggests a DNA-like fragmentation mechanism in tcDNA. The loss of nucleobases constitutes the second prevalent dissociation pathway observed in tcDNA. Comparison of partially and fully modified oligonucleotides indicates a pronounced impact of the sugar-moiety on the base loss. As this event initiates cleavage of the backbone, the presented results provide new mechanistic insights into the fragmentation of DNA in the gas-phase. The influence of the sugar-moiety on the dissociation extends to tcDNA:DNA and tcDNA:RNA hybrid duplexes, where base loss was found to be much more prominent from sugar-modified oligonucleotides than from their natural complements. Further prominent dissociation channels are strand separation and backbone cleavage of the single strands, as well as the ejection of backbone fragments from the intact duplex. The latter pathway depends noticeably on the base sequence. Moreover, it gives evidence of the high stability of the hybrid dimers, and thus directly reflects the affinity of tcDNA for its target in the cell. As the cellular target of tcDNA is a pre-mRNA, the structure was designed to discriminate RNA from DNA complements, which could be demonstrated by mass spectrometric experiments.

Decomposition studies of group 6 hexacarbonyl complexes. Part 1: Production and decomposition of Mo(CO)6 and W(CO)6

Chemical studies of superheavy elements require fast and efficient techniques, due to short half-lives and low production rates of the investigated nuclides. Here, we advocate for using a tubular flow reactor for assessing the thermal stability of the Sg carbonyl complex – Sg(CO)6. The experimental setup was tested with Mo and W carbonyl complexes, as their properties are established and supported by theoretical predictions. The suggested approach proved to be effective in discriminating between the thermal stabilities of Mo(CO)6 and W(CO)6. Therefore, an experimental verification of the predicted Sg–CO bond dissociation energy seems to be feasible by applying this technique. By investigating the effect of 104,105Mo beta-decay on the formation of 104,105Tc carbonyl complex, we estimated the lower reaction time limit for the metal carbonyl synthesis in the gas phase to be more than 100 ms. We examined further the influence of the wall material of the recoil chamber, the carrier gas composition, the gas flow rate, and the pressure on the production yield of 104Mo(CO)6, so that the future stability tests with Sg(CO)6 can be optimized accordingly.

Immune response in pemphigus and beyond: progresses and emerging concepts

Pemphigus vulgaris (PV) and pemphigus foliaceus (PF) are two severe autoimmune bullous diseases of the mucosae and/or skin associated with autoantibodies directed against desmoglein (Dsg) 3 and/or Dsg1. These two desmosomal cadherins, typifying stratified epithelia, are components of cell adhesion complexes called desmosomes and represent extra-desmosomal adhesion receptors. We herein review the advances in our understanding of the immune response underlying pemphigus, including human leucocyte antigen (HLA) class II-associated genetic susceptibility, characteristics of pathogenic anti-Dsg antibodies, antigenic mapping studies as well as findings about Dsg-specific B and T cells. The pathogenicity of anti-Dsg autoantibodies has been convincingly demonstrated. Disease activity and clinical phenotype correlate with anti-Dsg antibody titers and profile while passive transfer of anti-Dsg IgG from pemphigus patients' results in pemphigus-like lesions in neonatal and adult mice. Finally, adoptive transfer of splenocytes from Dsg3-knockout mice immunized with murine Dsg3 into immunodeficient mice phenotypically recapitulates PV. Although the exact pathogenic mechanisms leading to blister formation have not been fully elucidated, intracellular signaling following antibody binding has been found to be necessary for inducing cell-cell dissociation, at least for PV. These new insights not only highlight the key role of Dsgs in maintenance of tissue homeostasis but are expected to progressively change pemphigus management, paving the way for novel targeted immunologic and pharmacologic therapies.

Resilience as a correlate of acute stress disorder symptoms in patients with acute myocardial infarction

OBJECTIVES Myocardial infarction (MI) may be experienced as a traumatic event causing acute stress disorder (ASD). This mental disorder has an impact on the daily life of patients and is associated with the development of post-traumatic stress disorder. Trait resilience has been shown to be a protective factor for post-traumatic stress disorder, but its association with ASD in patients with MI is elusive and was examined in this study. METHODS We investigated 71 consecutive patients with acute MI within 48 h of having stable haemodynamic conditions established and for 3 months thereafter. All patients completed the Acute Stress Disorder Scale and the Resilience Scale to self-rate the severity of ASD symptoms and trait resilience, respectively. RESULTS Hierarchical regression analysis showed that greater resilience was associated with lower symptoms of ASD independent of covariates (b=-0.22, p<0.05). Post hoc analysis revealed resilience level to be inversely associated with the ASD symptom clusters of re-experiencing (b=-0.05, p<0.05) and arousal (b=-0.09, p<0.05), but not with dissociation and avoidance. CONCLUSIONS The findings suggest that patients with acute MI with higher trait resilience experience relatively fewer symptoms of ASD during MI. Resilience was particularly associated with re-experiencing and arousal symptoms. Our findings contribute to a better understanding of resilience as a potentially important correlate of ASD in the context of traumatic situations such as acute MI. These results emphasise the importance of identifying patients with low resilience in medical settings and to offer them adequate support.

Hypercoagulation and hyperkinetic blood pressure indicative of physiological loss-of-control despite behavioural control in Africans: The SABPA study.

OBJECTIVES A dissociation between behavioural (in-control) and physiological parameters (indicating loss-of-control) is associated with cardiovascular risk in defensive coping (DefS) Africans. We evaluated relationships between DefS, sub-clinical atherosclerosis, low-grade inflammation and hypercoagulation in a bi-ethnic sex cohort. METHODS Black (Africans) and white Africans (Caucasians) (n = 375; aged 44.6 ± 9.7 years) were included. Ambulatory BP, vascular structure (left carotid cross-sectional wall area (L-CSWA) and plaque counts), and markers of coagulation and inflammation were quantified. Ethnicity/coping style interaction was revealed only in DefS participants. RESULTS A hypertensive state, less plaque, low-grade inflammation, and hypercoagulation were more prevalent in DefS Africans (27-84%) than DefS Caucasians (18-41%). Regression analyses demonstrated associations between L-CSWA and 24 hour systolic BP (R(2) = 0.38; β = 0.78; p < 0.05) in DefS African men but not in DefS African women or Caucasians. No associations between L-CSWA and coagulation markers were evident. CONCLUSION Novel findings revealed hypercoagulation, low-grade inflammation and hyperkinetic BP (physiological loss-of-control responses) in DefS African men. Coupled to a self-reported in-control DefS behavioural profile, this reflects dissociation between behaviour and physiology. It may explain changes in vascular structure, increasing cerebrovascular disease risk in a state of hyper-vigilant coping.

Complex syntax in autistic spectrum disorders: A study with relative clauses

Background The few studies that have evaluated syntax in autism spectrum disorder (ASD) have yielded conflicting findings: some suggest that once matched on mental age, ASD and typically developing controls do not differ for grammar, while others report that morphosyntactic deficits are independent of cognitive skills in ASD. There is a need for a better understanding of syntax in ASD and its relation to, or dissociation from, nonverbal abilities. Aims Syntax in ASD was assessed by evaluating subject and object relative clause comprehension in adolescents and adults diagnosed with ASD with a performance IQ within the normal range, and with or without a history of language delay. Methods & Procedures Twenty-eight participants with ASD (mean age 21.8) and 28 age-matched controls (mean age 22.07) were required to point to a character designated by relative clauses that varied in syntactic complexity. Outcomes & Results Scores indicate that participants with ASD regardless of the language development history perform significantly worse than age-matched controls with object relative clauses. In addition, participants with ASD with a history of language delay (diagnosed with high-functioning autism in the DSM-IV-TR) perform worse on subject relatives than ASD participants without language delay (diagnosed with Asperger syndrome in the DSM-IV-TR), suggesting that these two groups do not have equivalent linguistic abilities. Performance IQ has a positive impact on the success of the task for the population with ASD. Conclusions & Implications This study reveals subtle grammatical difficulties remaining in adult individuals with ASD within normal IQ range as compared with age-matched peers. Even in the absence of a history of language delay in childhood, the results suggest that a slight deficit may nevertheless be present and go undetected by standardized language assessments. Both groups with and without language delay have a similar global performance on relative clause comprehension; however, the study also indicates that the participants with reported language delay show more difficulty with subject relatives than the participants without language delay, suggesting the presence of differences in linguistic abilities between these subgroups of ASD.

Variability of mesospheric water vapor above Bern in relation to the 27-day solar rotation cycle

Many studies investigated solar–terrestrial responses (thermal state, O₃ , OH, H₂O) with emphasis on the tropical upper atmosphere. In this paper the Focus is switched to water vapor in the mesosphere at a mid-latitudinal location. Eight years of water vapor profile measurements above Bern (46.88°N/7.46°E) are investigated to study oscillations with the Focus on periods between 10 and 50 days. Different spectral analyses revealed prominent features in the 27-day oscillation band, which are enhanced in the upper mesosphere (above 0.1 hPa, ∼64 km) during the rising sun spot activity of solar cycle 24. Local as well as zonal mean Aura MLS observations Support these results by showing a similar behavior. The relationship between mesospheric water and the solar Lyman-α flux is studied by comparing thesi-milarity of their temporal oscillations. The H₂O oscillation is negatively correlated to solar Lyman-α oscillation with a correlation coefficient of up to −0.3 to −0.4, and the Phase lag is 6–10 days at 0.04 hPa. The confidence level of the correlation is ≥99%. This finding supports the assumption that the 27-day oscillation in Lyman-α causes a periodical photo dissociation loss in mesospheric water. Wavelet power spectra, cross-wavelet transform and wavelet coherence analysis (WTC)complete our study. More periods of high common wavelet power of H₂O and solar Lyman-α are present when amplitudes of the Lyman-α flux increase. Since this is not a measure of physical correlation a more detailed view on WTC is necessary, where significant (two sigma level)correlations occur intermittently in the 27 and 13-day band with variable Phase lock behavior. Large Lyman-α oscillations appeared after the solar super storm in July 2012 and the H₂O oscillations show a well pronounced anticorrelation. The competition between advective transport and photo dissociation loss of mesospheric water vapor may explain the sometimes variable Phase relationship of mesospheric H₂O and solar Lyman-α oscillations. Generally, the WTC analysis indicates that solar variability causes observable photochemical and dynamical processes in the mid-latitude mesosphere.

Sulfur Isotopic Ratios at 67P/Churyumov-Gerasimenko and Characterization of ROSINA-DFMS FM & FS

Comets are thought to be the most pristine bodies present in the Solar System. In consequence of spending the majority of their existence beyond 30 AU, their composition can give insights on the physical and chemical conditions during their formation. Since August 2014 the European Space Agency spacecraft Rosetta accompanies the Jupiter family comet 67P/Churyumov-Gerasimenko on its way to perihelion and beyond. In this study the isotope fractionation of 34S are reported in H2S, OCS, SO2, S2, and CS2 at 67P. In addition for the first time the isotope fractionation for 33S is presented for cometary volatiles. The ratio 32S/33S is given for H2S, SO2 and a tentative value is given for CS2. With a mean value of -50 ± 22‰ and -306 ± 31‰ for δ34S and δ33S respectively, H2S shows a significant depletion in both 34S and 33S. For SO2 the depletion is less distinct with δ34S and δ33S being -67 ± 40‰ and -130 ± 53‰, respectively. The strongest depletion is present for CS2 with -114 ± 21‰and -276 ± 55‰, respectively. For OCS and S2 only δ34S could be determined which is -252 ± 77‰ and -357 ± 145‰, respectively. A comparison with sulfur isotopic ratios measured in SiC grains revealed that both SiC grains and the five volatile species have similar sulfur isotopic ratios. However, it is beyond the scope of this work to investigate the possibility of a link between SiC grains and cometary ices. Nevertheless, mass-dependent or mass-independent fractionation due to photo dissociation can be ruled out as sole cause of the seen depletion of 33S and 34S. Furthermore, an upper limit of (9.64 ± 0.19)·10.4 for D/H in HDS has been determined. This value is about a factor two higher than D/H in H2O for the same comet reported by (Altwegg et al., 2015). Besides the investigation concerning isotopic ratios of sulfur bearing species in this work the calibration and characterization of ROSINA/DFMS has been continued. Here it is reported about the deviation of the mass scale for MCP/LEDA low resolution spectra and the calibration measurements performed in the laboratory. Furthermore the outcome of the attempt to describe the sensitivity of DFMS with an empirical function will be discussed. The last part of the characterization of DFMS is dedicated to determine the so-called individual pixel gain for the laboratory and the flight model. Moreover, correlation between the depletion’s manifestation of the MCP with respect to the applied voltages has been investigated for both models. It has been found that further measurements are needed to understand the manifestation of depletion at the laboratory model. For the model on board of Rosetta it could be shown that most of the present feature are due to the usage of the MCP and suggestions have been made in order to answer the remaining question considering the depletion of the MCP.

Religion as Culture? How European and American High Courts Endorse Majority Religion

This paper examines a trend in European and American High Courts to endorse majority religion by transforming it into “culture”, and thus by secularizing it. To dissociate religion and state is the hallmark of the liberal state. However, no state has ever managed a perfect separation, not even the American. Under conditions of mounting religious pluralism and ongoing secularization, there is pressure on the state to live up to its “neutrality”. A main strategy to square the circle of neutrality and incomplete dissociation from religion is to declare it “culture”, which gives the state the license to associate or even identify with it (as guardian of nationhood). The paper compares recent American and European High Court rules on religious symbols (especially crucifixes) that exhibits this strategy, addressing similarities and differences as well as the limits and pitfalls of “culturalizing” religion.

Effect of Nontronite Smectite Clay on the Chemical Evolution of Several Organic Molecules under Simulated Martian Surface Ultraviolet Radiation Conditions

Most of the phyllosilicates detected at the surface of Mars today are probably remnants of ancient environments that sustained long-term bodies of liquid water at the surface or subsurface and were possibly favorable for the emergence of life. Consequently, phyllosilicates have become the main mineral target in the search for organics on Mars. But are phyllosilicates efficient at preserving organic molecules under current environmental conditions at the surface of Mars? We monitored the qualitative and quantitative evolutions of glycine, urea, and adenine in interaction with the Fe3+-smectite clay nontronite, one of the most abundant phyllosilicates present at the surface of Mars, under simulated martian surface ultraviolet light (190-400 nm), mean temperature (218 +/- 2 K), and pressure (6 +/- 1 mbar) in a laboratory simulation setup. We tested organic-rich samples that were representative of the evaporation of a small, warm pond of liquid water containing a high concentration of organics. For each molecule, we observed how the nontronite influences its quantum efficiency of photodecomposition and the nature of its solid evolution products. The results reveal a pronounced photoprotective effect of nontronite on the evolution of glycine and adenine; their efficiencies of photodecomposition were reduced by a factor of 5 when mixed at a concentration of 2.6x10(-2) mol of molecules per gram of nontronite. Moreover, when the amount of nontronite in the sample of glycine was increased by a factor of 2, the gain of photoprotection was multiplied by a factor of 5. This indicates that the photoprotection provided by the nontronite is not a purely mechanical shielding effect but is also due to stabilizing interactions. No new evolution product was firmly identified, but the results obtained with urea suggest a particular reactivity in the presence of nontronite, leading to an increase of its dissociation rate. Key Words: Martian surface-Organic chemistry-Photochemistry-Astrochemistry-Nontronite-Phyllosilicates. Astrobiology 15, 221-237.

BIOLOGICAL AND BIOCHEMICAL CHARACTERIZATION OF MURINE-TUMOR SPECIFIC TRANSPLANTATION ANTIGENS (ISOTACHOPHORESIS, IMMUNOGENICITY)

Tumor-specific transplantation antigens (TSTA) are individually distinct neoantigens expressed on the cells of chemically-induced neoplasms. TSTA are operationally defined by immunization of syngeneic mice against challenge with viable tumor cells. Immunization with cell surface or extracted TSTA induces specific resistance to transplanted tumor cells. The biological and biochemical nature of TSTA was investigated in the 3-methylcholanthrene-induced fibrosarcomas of female C3H/HeJ mice, MCA-F and MCA-D. Tumor cell suspensions were extracted by treatment with 3M KCl or 2.5% butanol solutions and the TSTA was partially purified by preparative isoelectric focusing. The isoelectric pH of TSTA purified from 3M KCl extracts was 5.8-6.0, and from butanol extracts was 6.4-6.6. Whereas immunization with 10('5) and 10('6) irradiated tumor cells induces complete rejection of tumor cell challenge over a two-fold-log dose range, immunization with ug quantities within a one-fold-log dose range of extracted TSTA induces only partial resistance to tumor challenge. Reduced immunogenicity of extracted TSTA is hypothesized to result from immunization of mice with insufficiently purified TSTA preparations. The hypothesis predicts that immunization with highly purified TSTA, free from interfering substances, induces complete rejection of tumor challenge over a broad dose range. To test the hypothesis preparative isotachophoresis (pITP) was used to purify TSTA from electrofocused TSTA fractions. Significant purification was achieved, as immunization with 15 pg to 1.5 ug (5 logs) of pITP-purified TSTA extracted from the MCA-F, or with 1 pg to 10 ng (4 logs) of TSTA from the MCA-D tumor induced specific resistance to tumor challenge. Despite 50,000 fold purification of TSTA, immunization induced partial, not complete, rejection of transplanted tumor cells. This suggests a clear dissociation of the immunogenicity and purification of extracted TSTA, indicating that the induction of partial immunity to tumor challenge is an intrinsic property of extracted TSTA.^

THE EQUILIBRIUM CONSTANT FOR THE GLYCINE SYNTHASE REACTION (ONE-CARBON, METABOLISM, FOLATE)

The equilibrium constant (K(,c)) under physiological conditions (38(DEGREES)C, 0.25 M ionic strength (I), pH 7.0) for the glycine synthase (GS) reaction (E C 2.1.2.1.0) (Equation 1) has been determined. (UNFORMATTED TABLE FOLLOWS)^ 5,10-CH(,2)-H(,4)Folate NADH NH (,4)+ CO(,2) ^ K(,c) = Eq. 1^ H(,4)Folate NAD('+) GLY ^(TABLE ENDS)^ The enzymatic instability of the GS enzyme complex itself has made it necessary to determine the overall K(,c) from the product of constants for the partial reactions of GS determined separately under the same conditions. The partial reactions are the H(,4)Folate-formaldehyde (CH(,2)(OH)(,2)) condensation reaction (Reaction 1) the K(,c) for which has been reported by this laboratory (3.0 x 10('4)), the lipoate (LipS(,2)) dehydrogenase reaction (LipDH) (Reaction 2) and the Gly-Lip^ decarboxylase reaction (Reaction 3) forming reduced lipoate (Lip(SH)(,2)), NH(,4)('+), CO(,2) and CH(,2)(OH)(,2.) (UNFORMATTED TABLE FOLLOWS)(,)^ H(,4)Fote + CH(,2)(OH)(,2) 5,10-CH(,2)-H(,4)Folate (1)^ Lip(SH)(,2) + NAD('+) LipS(,2) + NADH + H('+) (2)^ H('+) + Gly + LipS(,2) Lip(SH)(,2) + NH(,4)('+) CO(,2) + CH(,2)(OH)(,2) (3)^(TABLE ENDS)^ In this work the K(,c) for Reactions 2 and 3 are reported.^ The K(,c)' for the LipDH reaction described by other authors was reported with unexplainable conclusions regarding the pH depend- ence for the reaction. These conclusions would imply otherwise unexpected acid dissociation constants for reduced and oxidized lipoate. The pK(,a)',s for these compounds have been determined to resolve discrepancy. The conclusions are as follows: (1) The K(,c) for the LipDH reaction is 2.08 x 10('-8); (2) The pK(,a)',s for Lip(SH)(,2) are 4.77(-COOH), 9.91(-SH), 11.59(-SH); for LipS(,2) the carboxyl pK(,a)' is 4.77; (3) Contrary to previous literature, the log K(,c)' for the LipDH reaction is a linear function of the pH, a conclusion supported by the values for the dissociation constants.^ The K(,c) for Reaction 3 is the product of constants for Reactions 4-7. (UNFORMATTED TABLE FOLLOWS)^ LipSHSCH(,2)OH + H(,2)O Lip(SH)(,2) + CH(,2)(OH)(,2) (4)^ H(,2)O + LipSHSCH(,2)NH(,3)('+) LipSHSCH(,2)OH + NH(,4)('+) (5)^ LipSHSCH(,2)NH(,2) + H('+) LipSHSCH(,2)NH(,3)('+) (6)^ Gly + LipS(,2) LipSHSCH(,2)NH(,2) + CO(,2) (7)^(TABLE ENDS)^ Reactions 4-6 are non-enzymatic reactions whose constants were determined spectrophotometrically. Reaction 7 was catalyzed by the partially purified P-protein of GS with equilibrium approached from both directions. The value for K(,c) for this reaction is 8.15 x 10('-3). The combined K(,c) for Reactions 4-7 or Reaction 3 is 2.4 M.^ The overall K(,c) for the GS reaction determined by combination of values for Reactions 1-3 is 1.56 x 10('-3). ^

A novel mechanism for regulation of calmodulin signaling by RC3

RC3, also known as neurogranin, is a small neuronal IQ domain protein whose only known function is to bind calmodulin (CaM). The hypothesis tested in this work was that RC3 alters the dynamics of the interaction of Ca 2+-CaM with CaM-kinase II, so that there is less CaM-kinase II activation for a given Ca2+ stimulus. To evaluate this hypothesis, we investigated the affinity and kinetics of the interactions of CaM with Ca 2+, RC3 and CaM-kinase II. We quantitated the interaction of the four CaM-kinase II isoforms with CaM and found that the KD for binding of CaM to CaM-kinase II ranged from 7 nM to 60 nM. Using stopped-flow fluorimetry, we determined the kinetics of the interaction of Ca2+-CaM with αCaM-kinase II, and found that the association rate constant is 2.1 × 10 M −1s−1 and the dissociation rate constant is 1.6 s−1. We investigated the effects of RC3 and αCaM-kinase II on the affinity of CaM for Ca2+ and found that both proteins alter the rate of dissociation of Ca2+ from CaM. RC3 increases the rate of dissociation of Ca2+ from the C-terminal binding sites of CaM from 9 s−1 to ∼500 s−1 , while αCaM-kinase II causes a decrease in the rate of dissociation from all four Ca2+ binding sites. Measurement of the rate of dissociation of Ca2+ from CaM in the presence of both RC3 and αCaM-kinase II revealed a role for RC3 in accelerating the dissociation of the Ca 2+-CaM-αCaM-kinase II complex at the end of a Ca2+ signal. We characterized the interaction of RC3 with apo-CaM and Ca 2+-CaM and found that the KD for both of these interactions is about 1 μM. We also directly tested whether RC3 slowed the dynamics of the binding of CaM to αCaM-kinase II and found that RC3 had no effect for large changes in Ca2+, and a modest effect for small changes in Ca2+ levels. Our overall conclusion is that the ability of RC3 to alter the interaction of Ca2+ with CaM allows RC3 to alter the dynamics of interaction of CaM with Ca2+-dependent targets such as CaM-kinase II. ^

The in vivo and in vitro formation of sticky DNA and the GAA.TTC trinucleotide repeat associated with Friedreich's ataxia

Friedreich's ataxia is caused by the expansion of the GAA•TTC trinucleotide repeat sequence located in intron 1 of the frataxin gene. The long GAA•TTC repeats are known to form several non-B DNA structures including hairpins, triplexes, parallel DNA and sticky DNA. Therefore it is believed that alternative DNA structures play a role in the loss of mRNA transcript and functional frataxin protein in FRDA patients. We wanted to further elucidate the characteristics for formation and stability of sticky DNA by evaluating the structure in a plasmid based system in vitro and in vivo in Escherichia coli. The negative supercoil density of plasmids harboring different lengths of GAA•TTC repeats, as well as either one or two repeat tracts were studied in E. coli to determine if plasmids containing two long tracts (≥60 repeats) in a direct repeat orientation would have a different topological effect in vivo compared to plasmids that harbored only one GAA•TTC tract or two tracts of < 60 repeats. The experiments revealed that, in fact, sticky DNA forming plasmids had a lower average negative supercoil density (-σ) compared to all other control plasmids used that had the potential to form other non-B DNA structures such as triplexes or Z-DNA. Also, the requirements for in vitro dissociation and reconstitution of the DNA•DNA associated region of sticky DNA were evaluated. Results conclude that the two repeat tracts associate in the presence of negative supercoiling and MgCl 2 or MnCl2 in a time and concentration-dependent manner. Interaction of the repeat sequences was not observed in the absence of negative supercoiling and/or MgCl2 or in the presence of other monovalent or divalent cations, indicating that supercoiling and quite specific cations are needed for the association of sticky DNA. These are the first experiments studying a more specific role of supercoiling and cation influence on this DNA conformation. To support our model of the topological effects of sticky DNA in plasmids, changes in sticky DNA band migration was measured with reference to the linear DNA after treatment with increasing concentrations of ethidium bromide (EtBr). The presence of independent negative supercoil domains was confirmed by this method and found to be segregated by the DNA-DNA associated region. Sequence-specific polyamide molecules were used to test the effect of binding of the ligands to the GAA•TTC repeats on the inhibition of sticky DNA. The destabilization of the sticky DNA conformation in vitro through this binding of the polyamides demonstrated the first conceptual therapeutic approach for the treatment of FRDA at the DNA molecular level. ^ Thus, examining the properties of sticky DNA formed by these long repeat tracts is important in the elucidation of the possible role of sticky DNA in Friedreich's ataxia. ^

Functional differences of beta-tubulin isotypes: A study of microtubule assembly and antimitotic drug resistance

Mammalian cells express 7 β-tubulin isotypes in a tissue specific manner. This has long fueled the speculation that different isotypes carry out different functions. To provide direct evidence for their functional significance, class III, IVa, and VI β-tubulin cDNAs were cloned into a tetracycline regulated expression vector and stably transfected Chinese hamster ovary cell lines expressing different levels of ectopic β-tubulin were compared for effects on microtubule organization, microtubule assembly and sensitivity to antimitotic drugs. It was found that all three isotypes coassembled with endogenous β-tubulin. βVI expression caused distinct microtubule rearrangements including microtubule dissociation from the centrosome and accumulation at the cell periphery; whereas expression of βIII and βVIa caused no observable changes in the interphase microtubule network. Overexpression of all 3 isotypes caused spindle malformation and mitotic defects. Both βIII and βIVa disrupted microtubule assembly in proportion to their abundance and thereby conferred supersensitivity to microtubule depolymerizing drugs. In contrast, βVI stabilized microtubules at low stoichiometry and thus conferred resistance to many microtubule destabilizing drugs but not vinblastine. The 3 isotypes caused differing responses to microtubule stabilizing drugs. Expression of βIII conferred paclitaxel resistance while βVI did not. Low expression of βIVa caused supersensitivity to paclitaxel, whereas higher expression resulted in the loss of supersensitivity. The results suggest that βIVa may possess an enhanced ability to bind paclitaxel that increases sensitivity to the drug and acts substoichiometrically. At high levels of βVIa expression, however, microtubule disruptive effects counteract the assembly promoting pressure exerted by increased paclitaxel binding, and drug supersensitivity is lost. From this study, I concluded that β-tubulin isotypes behave differently from each other in terms of microtubule organization, microtubule assembly and dynamics, and antimitotic drug sensitivity. The isotype composition of cell can impart subtle to dramatic effects on the properties of microtubules leading to potential functional consequences and opening the opportunity to exploit differences in microtubule isotype composition for therapeutic gain. ^