Radiation damage to a DNA-binding protein. Combined circular dichroism and molecular dynamics simulation analysis.

The E. coli lactose operon, the paradigm of gene expression regulation systems, is the best model for studying the effect of radiation on such systems. The operon function requires the binding of a protein, the repressor, to a specific DNA sequence, the operator. We have previously shown that upon irradiation the repressor loses its operator binding ability. The main radiation-induced lesions of the headpiece have been identified by mass spectrometry. All tyrosine residues are oxidized into 3,4-dihydroxyphenylalanine (DOPA). In the present study we report a detailed characterization of the headpiece radiation-induced modification. An original approach combining circular dichroism measurements and the analysis of molecular dynamics simulation of headpieces bearing DOPA-s instead of tyrosines has been applied. The CD measurements reveal an irreversible modification of the headpiece structure and stability. The molecular dynamics simulation shows a loss of stability shown by an increase in internal dynamics and allows the estimation of the modifications due to tyrosine oxidation for each structural element of the protein. The changes in headpiece structure and stability can explain at least in part the radiation-induced loss of binding ability of the repressor to the operator. This conclusion should hold for all proteins containing radiosensitive amino acids in their DNA-binding site.

Molecular dynamics simulation for probing the flexibility of the 35 nucleotide SL1 sequence kissing complex from HIV-1Lai genomic RNA.

The SL1 stem-loop located in the encapsidation domain is responsible for initiating the dimerisation of HIV-1 genomic RNA by means of a loop-loop interaction known as Kissing Complex (KC). The SL1 secondary structure has been predicted as a 35 nucleotides [K. G. Murti, M. Bondurant, and A. Tereba. J Virol 37, 411-419 (1981)] stem-loop composed of a 4 base pairs (bp) terminal duplex, a 4 nt asymmetrical internal loop, a 7 bp internal duplex, and a 9 nt apical loop. Several high resolution structures of the monomer and of KC of a 23 nt sequence containing only the internal duplex and the apical loop of SL1 are available in the literature. No experimental high resolution structure of the complete native SL1 sequence has been reported so far, either for the monomer or for KC. The asymmetrical internal loop has been described from NMR studies of different monomeric hairpin sequences, leading to divergent results, which suggests its high flexibility. In this work, we built a SL1(35) KC model which was submitted to a 31 ns molecular dynamics simulation (MD). Our results allows to describe the internal dynamics of SL1(35) KC and the differences of behavior of the different parts of the dimer. Thus, we could show the stability of the interactions between the two apical loops and of the terminal duplexes, the destabilization of the internal duplexes and the high flexibility of the asymmetrical internal loops.

Conformational pathway for the kissing complex-->extended dimer transition of the SL1 stem-loop from genomic HIV-1 RNA as monitored by targeted molecular dynamics techniques.

HIV-1 retroviral genomic RNA dimerization is initiated by loop-loop interactions between the SL1 stem-loops of two identical RNA molecules. The SL1-SL1 unstable resulting kissing complex (KC) then refolds irreversibly into a more stable complex called extended dimer (ED). Although the structures of both types of complex have been determined, very little is known about the conformational pathway corresponding to the transition, owing to the difficulty of observing experimentally intermediate conformations. In this study, we applied targeted molecular dynamics simulation techniques (TMD) to the phosphorus atoms for monitoring this pathway for the backbone, and a two-step strategy was adopted. In a first step, called TMD(-1), the dimer structure was constrained to progressively move away from KC without indicating the direction, until the RMSD from KC reaches 36A. A total of 20 TMD(-1) simulations were performed under different initial conditions and different simulation parameters. For RMSD ranging between 0 and 22A, the whole set of TMD(-1) simulations follows a similar pathway, then divergences are observed. None of the simulations leads to the ED structure. At RMSD=22A, the dimers look like two parallel Us, still linked by the initial loop-loop interaction, but the strands of the stems (the arms of the Us) are positioned in such a manner that they can form intramolecular as well as intermolecular Watson-Crick base-pairs. This family of structure is called UU. In a second step (TMD simulations), 18 structures were picked up along the pathways generated with TMD(-1) and were constrained to move toward ED by decreasing progressively their RMSD from ED. We found that only structures from the UU family are able to easily reach ED-like conformations of the backbones without exhibiting a large constraint energy.

DOC-2/hDab2, a candidate tumor suppressor gene involved in the development of gestational trophoblastic diseases.

Gestational trophoblastic diseases comprise a spectrum of interrelated diseases including partial mole, complete mole and gestational choriocarcinoma. Using reverse transcriptase PCR (RT-PCR) analysis, we identified higher levels of DOC-2/hDab2 expression in the normal trophoblast cells in culture than in choriocarcinoma cell lines. Subsequent study using immunohistochemistry showed high levels of DOC-2/hDab2 protein expression in normal trophoblast tissues but significantly lower levels of expression in gestational trophoblastic disease tissues, particularly in complete mole and choriocarcinoma. When DOC-2/hDab2 was transfected into the choriocarcinoma cell lines, Jar, JEG and BeWo, the stable transfectants showed significantly reduced growth rate in culture. These data suggest that down regulation of DOC-2/hDab2 may play an important role in the development of gestational trophoblastic diseases.

Fluorescence anisotropy decay due to rotational brownian motion of ethidium intercalated in double strand DNA.

The transient fluorescence of solutions of ethidium bromide . DNA complexes has been measured by pulse fluorimetry at different temperatures and in solvents containing various amounts of sucrose. The molar ratio of ethidium to nucleotides was low. Under these conditions the anisotropy decay was due to the Brownian motion of ethidium molecules intercalated in the double strand DNA molecules. This anisotropy decay could be described by a sum of 3 exponential terms, with correlation times 01, 02, 03 which were linear functions of the ratio of the solvent viscosity to the absolute temperature (n/T). The amplitude of the exponential term characterized by the shortest correlation time (01) has been found to depend on temperature while the ratio of the amplitude of the two other terms (characterized by 02 and 03) was independent of temperature. These results were interpreted as follows: 01 corresponds to a fast motion of the dye in its site. 02 and 03 describe a tortional motion of the ethidium bromide. DNA complex, involving several nucleotide pairs.

X-ray diffuse scattering and rigid-body motion in crystalline lysozyme probed by molecular dynamics simulation.

Rigid-body motions are determined from a 1 ns molecular dynamics simulation of the unit cell of orthorhombic hen egg-white lysozyme and their contribution to X-ray diffuse scattering intensities are examined. Using a dynamical cluster technique, groups of backbone atoms that move as approximately rigid bodies are derived from the intramolecular interatomic fluctuation matrix. These groups tend to be local in the sequence or connected by disulphide bonds, and contain on average five residues each, X-ray diffuse scattering patterns, which are sensitive to collective motions, are calculated from the full simulation trajectory (including all the protein degrees of freedom). The results reproduce the main features of the experimental scattering. Diffuse scattering is also calculated from fitted trajectories of the rigid bodies. The full simulation diffuse scattering and atomic displacements are found to be well reproduced by a model in which the backbone atoms form the rigid groups determined using the dynamical cluster technique and the individual side-chains behave as separate rigid bodies: the resulting R-factor with the full simulation scattering is 5%. Quantitatively poorer agreement is obtained from trajectories in which the secondary structural elements of the protein are considered rigid. Rigid whole-molecule and domain motions make only minor contributions to the protein atom displacements. Finally, correlations in the interatomic fluctuations are examined directly using a canonical method.

From atomic to mesoscopic descriptions of the internal dynamics of DNA.

An analysis of four 1-ns molecular dynamics trajectories for two different 15-bp oligonucleotides is presented. Our aim is to show which groups of atoms can be treated as rigid bodies within a bead representation of DNA, independently of the base sequence and for any conformations belonging to the A/B family. Five models with moderate intragroup deformations are proposed in which the groups are formed of atoms belonging to a single nucleotide or to a complementary nucleotide pair. The influence of group deformation in two of these models is studied using canonical correlation analysis, and it is shown that the internal DNA dynamics is indeed dominated by the rigid motion of the defined atom groups. Finally, using one of the models within a bead representation of duplex DNA makes it possible to obtain stretching, torsional, and bending rigidities in reasonable agreement with experiment but points to strongly correlated stretching motions.

An interpretation of the binding of ethidium bromide to the core nucleosome, based on Monte Carlo calculations.

We propose a model of ethidium binding to the nucleosome core particle. Ligand molecules are assumed to intercalate between the base pairs of the DNA core particle. The DNA is assumed to be divided into a finite number of segments. In the native core particle, one segment only is accessible to ethidium. The other DNA segments become progressively accessible when the binding ratio is increased. The binding isotherm resulting from such a model has been determined by a Monte Carlo method. The computed Scatchard plot exhibits the shape characteristic of a cooperative process. By fitting this curve with the experimental data of Erard et al. [1] one finds that 130 over the 140 DNA base pairs are accessible to the ethidium binding. The full accessibility of these sites is obtained when 8 or 9 ethidium at least are already bound to the core particle.

A method for deciding whether two experimental fluorescence anisotropy decay curves are significantly different.

We describe a method to compare two fluorescence anisotropy decay curves. After numerical deconvolution of both decays by a non a priori method [1], their difference, D, is considered. The variance is computed for each point of D. A confidence interval is defined which allows a decision to be made as to the significance of D. Information on the time range in which the changes of the fluorescence anisotropy decay occur is directly available. This analysis is particularly well suited for following the perturbations induced by an effector. It has been tested on 3-phosphoglycerate kinase in the presence and in the absence of ATP and 3-phosphoglycerate. We consider that this method leads to a significant improvement in the application of time resolved depolarization experiments.

Fluorescence anisotropy decay of ethidium bromide bound to nucleosomal core particles.

We have measured the fluorescence anisotropy decay of ethidium bromide bound to nucleosomal core particles (145 DNA base pairs) for very small values of the binding ratio (0.0005 less than or equal to r less than or equal to 0.01). For r = 0.0005 the anisotropy decay could be described by a sum of two exponential functions. The two correlation times theta 1 and theta 2 increase with r until r congruent to 0.0025 and then decrease while the apparent fundamental anisotropy A'0 decreases until r congruent to 0.0025 and then remains constant. The anisotropy decay parameters of the first ethidium molecule bound to a core particle have been obtained by extrapolating theta 1, theta 2 and A'0 to r = 0. We propose the following interpretation of these results. The first bound ethidium molecule is located on a DNA segment linked by its two ends to the histone core. This ethidium molecule follows the torsional motion of the DNA segment. The length of this segment (15 base pairs) was determined by fitting a mathematical expression, derived from the torsional dynamics of DNA, to the extrapolated anisotropy decay. The second ethidium molecule binds to the same DNA segment which explains the decrease of A'0 by fast excitation energy transfer. At the same time theta 1 and theta 2 increase. On binding, a third ethidium molecule breaks the links between the DNA segment and the histone core. This entails the decrease of theta 1 and theta 2.

Thermal stability of the Z conformation of the hexanucleoside pentaphosphate d(br5CGbr5CGbr5CG): evidence for a conformational transition before melting.

The thermal stability of the hexanucleoside pentaphosphate d(br5CGbr5CGbr5CG) has been studied at two nucleotide concentrations, in the presence of 1 M NaClO4. At low nucleotide concentration (7 X 10(-5) M), circular dichroism experiments show a conformational transition from the Z conformation to another conformation, named X, which is not the B conformation, as the temperature is increased from 0 to 35 degrees C. Between 40 and 65 degrees C, another transition is observed which corresponds to the melting of the X conformation. At higher nucleotide concentration (2 X 10(-3) M), circular dichroism and 31P nuclear magnetic resonance experiments show that at low temperature (br5dC-dG)3 adopts the Z conformation. There are associations between the oligonucleotides which progressively disappear as the temperature increases. In the range 35-60 degrees C a transition from the Z conformation to another conformation is observed. This new conformation is the X conformation detected at low nucleotide concentration.

Discrimination of complete hydatidiform mole from its mimics by immunohistochemistry of the paternally imprinted gene product p57KIP2.

The p57KIP2 protein is a cell cycle inhibitor and tumor suppressor encoded by a strongly paternally imprinted gene. We explored the utility of p57KIP2 as a diagnostic marker in hydatidiform mole, a disease likely the result of abnormal dosage and consequent misexpression of imprinted genes. Using a monoclonal antibody on paraffin-embedded, formalin-fixed tissue sections, the authors evaluated p57KIP2 expression in normal placenta and in 149 gestations including 59 complete hydatidiform moles, 39 PHMs, and 51 spontaneous losses with hydropic changes. p57KIP2 was strongly expressed in cytotrophoblast and villous mesenchyme in normal placenta, all cases of partial hydatidiform moles (39 of 39) and all spontaneous losses with hydropic changes (51 of 51). In contrast, p57KIP2 expression in cytotrophoblast and villous mesenchyme was absent or markedly decreased in 58 of 59 complete hydatidiform moles. In all gestations p57KIP2 was strongly expressed in decidua and in intervillous trophoblast islands, which served as internal positive controls for p57KIP2 immunostaining. p57KIP2 immunohistochemistry can reliably identify most cases of complete hydatidiform mole irrespective of gestational age and is thus a useful diagnostic adjunct, complementary to ploidy analysis, in the diagnosis of hydatidiform mole.

Fetal vasculitis in preterm newborns: interrelationships, modifiers, and antecedents.

Histologic expressions of the fetal inflammatory response predict preterm delivery and neonatal disorders. We examined 1146 placentas in the Developmental Epidemiology Network data set for histologic evidence of membrane inflammation (subchorionitis, chorionitis, and chorioamnionitis) and fetal vasculitis (acute umbilical vasculitis or chorionic vasculitis). Our main findings are that (1) in the presence of membrane inflammation, fetal vasculitis is common, (2) duration of membrane rupture and gestational age appear to modify the risk of fetal vasculitis, (3) this risk modification differs for the different components of fetal vasculitis, i.e. umbilical and chorionic vasculitis, and (4) antecedents can be identified that appear to increase or decrease the risk of fetal vasculitis among births with membrane inflammation. We conclude that fetal vasculitis, the morphologic component of the fetal inflammatory response, might not be a homogeneous entity and deserves further study.

Downregulation of placental syncytin expression and abnormal protein localization in pre-eclampsia.

Development of placentation and successful pregnancy depend on co-ordinated interactions between the maternal decidua and myometrium, and the invasive properties of the fetal trophoblast. Syncytin, a protein encoded by the envelope gene of a recently identified human endogenous defective retrovirus, HERV-W, is highly expressed in placental tissue. Previously, we have shown that the major site of syncytin expression is the placental syncytiotrophoblast, a fused multinuclear syncytium originating from cytotrophoblast cells. Here we present the first evidence that in pre-eclampsia, syncytin gene expression levels are dramatically reduced. Additionally, immunohistochemical examination of normal placentae and placentae from women with pre-eclampsia reveals that the syncytin protein in placental tissue from women with pre-eclampsia is localized improperly to the apical syncytiotrophoblast microvillous membrane as opposed to its normal location on the basal syncytiotrophoblast cytoplasmic membrane. Our previous results suggest that syncytin may mediate placental cytotrophoblast fusion in vivo and may play an important role in human placental morphogenesis. The present study suggests that altered expression of the syncytin gene, and altered cellular location of its protein product, may contribute to the aetiology of pre-eclampsia.

Early complete hydatidiform moles contain inner cell mass derivatives.

In four cases of early complete hydatidiform moles, confirmed to be androgenetic in origin by DNA studies, we have identified nonchorionic inner cell mass derived structures which are not commonly observed in specimens of later gestational age. These structures include nucleated red blood cells, endothelial cells, stromal macrophages, amnion and yolk sac. The latter four structures were confirmed by specific immunocytochemical stains. Recognition that such structures can accompany complete hydatidiform moles has both theoretical and practical significance. From a theoretical perspective, it demonstrates that the maternal genome is not required for the initiation of amniogenesis, development of the yolk sac, vasculogenesis, or hematopoiesis. From a practical perspective it emphasizes that complete hydatidiform moles, with their markedly increased risk of subsequent choriocarcinoma, cannot be excluded based on the finding of "fetal structures."

Cardiac histologic pathology characteristic of trisomies 13 and 21.

To identify fetal histologic features characteristic of specific chromosomal anomalies, we reviewed histologic slides of 415 cases, including therapeutic and spontaneous abortuses, stillbirths, and perinatal deaths. These included 126 cases (30%) with karyotypically confirmed trisomy 21 and 23 cases (5.5%) with trisomy 13. Two histologic abnormalities of the fetal heart were identified that correlated with specific karyotypic abnormalities: (1) a discrete central papillary muscle calcification was present in 14 of 85 (16%) cases with trisomy 21, in seven of 18 (39%) cases with trisomy 13, and in six of 255 (2%) controls (P less than .001); and (2) a focal ventricular epicardial lymphocytic infiltrate was present in 22 of 93 (24%) cases with trisomy 21 versus nine of 284 (3%) controls (P less than .001). When both histologic abnormalities coexisted, trisomy 21 was present in five of six cases (83%). Neither histologic finding was significantly associated with fetal or maternal infection or congenital heart defects. In a restricted prospective study of the hearts of fetuses with trisomy 21, papillary muscle calcification was demonstrated by specimen radiographs in four of six (67%) cases; one case was studied by specimen ultrasonogram, which identified a papillary muscle echodensity. We conclude that (1) a focal ventricular epicardial lymphocytic infiltrate is characteristic of trisomy 21, (2) papillary muscle microcalcifications are characteristic of trisomies 13 and 21, and (3) further studies are needed to determine whether papillary muscle calcification might be useful in antenatal ultrasonographic screening for chromosomal anomalies.

Absence of normal-appearing proximal tubules in the fetal and neonatal kidney: prevalence and significance.

Rare fetuses and neonates with kidneys lacking normal-appearing proximal tubules have been described. In order to ascertain the prevalence of this histologic finding, and to study its associated clinicopathologic features, 500 consecutive perinatal autopsies (performed from 1981 to 1985) were reviewed. Kidneys lacking normal-appearing proximal tubules were found in six of 500 (1.2%) perinatal autopsies (one liveborn and five stillborn cases). The liveborn infant was one of a sibship with renal tubular dysgenesis. Four of the stillborn fetuses were derived from monochorionic twin gestations; the histologic abnormality was present in only one fetus from each twin pair. Three of the four twin pairs had pathologic features suggestive of twin-to-twin transfusion, with the renal abnormality present in the donor twin; renal hypoplasia existed in two instances. The fourth affected twin was a stillborn acardiac fetus with multiple congenital anomalies and unilateral renal agenesis. The fifth stillborn was a hydropic fetus with trisomy 21 and renal hypoplasia. In this series, lack of recognizable renal proximal tubules most often was not a manifestation of renal tubular dysgenesis. The histologic finding was associated with stillborn, renal hypoplasia, and congenital anomalies, and was strongly associated with monochorionic twinning (P = 0.001). In the stillborn cases in this series, we suggest that this finding may represent renal tubular degeneration resulting from renal hypoperfusion.

Localized endometrial proliferations associated with pregnancy: clinical and histopathologic features of 11 cases.

Eleven cases of an unusual endometrial glandular proliferation associated with early pregnancy are reported. All lesions were incidental discoveries in first-trimester gestational endometria (two elective abortions; five spontaneous abortions; three hydatidiform moles; one tubal ectopic pregnancy). Most patients (nine of 11; 82%) were older than 30 years of age; associated clinical features included oligoovulation (two), hypertension (one), and obesity (one). All lesions were small and localized, and displayed similar histological features of variable severity including glandular expansion with smooth external contours; epithelial stratification (4 to 15 layers); cribriforming (focal to extensive); mitotic activity; bland nuclear cytology; and prominent intraglandular calcifications (eight cases; 72%). Although the natural history of these distinctive pregnancy-associated endometrial lesions was unknown, nine lesions were initially classified as benign, and two were interpreted as atypical endometrial hyperplasia or focal adenocarcinoma. Follow-up for an average of 34 months (range, 18 to 56) in nine patients showed no residual endometrial lesion (seven endometrial curettages and two hysterectomies). Three patients followed by curettage have subsequently completed successful pregnancies. This unusual lesion may represent a localized, endometrial proliferation induced by pregnancy; although some endometrial lesions may display striking architectural complexity, follow-up to date suggests a benign behavior.

Atypical immature metaplastic-like proliferations of the cervix: diagnostic reproducibility and viral (HPV) correlates.

Although some immature squamous lesions (papillary immature metaplasias) of the cervix have been described and associated with human papillomaviruses (HPV), nonpapillary atypical immature squamous proliferations (AISPs) are a poorly defined entity and range from atypical reactive metaplasias to squamous intraepithelial lesions resembling immature metaplasia. This study examined the diagnostic reproducibility of AISPs and their relationship to HPV nucleic acids. Forty-four diagnostically problematic AISPs were studied. Based on nuclear density (crowding), chromasia, variation (anisokaryosis) in nuclear size, and surface cytoplasmic maturation, cases were independently scored by 2 observers as (1) probably reactive (Rx), (2) not otherwise specified (NOS), and (3) squamous intraepithelial lesion (SIL). Extracted archival DNA was scored for HPV by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. Interobserver reproducibility (kappa statistic) and HPV correlates (chi square) were computed. Approximately one third of cases were classified in each category by the observers. Interobserver reproducibility was excellent (0.80), poor (0.23), and fair to good (0.41) for a diagnosis of Rx, NOS, and SIL, respectively. Differences in HPV DNA positivity between Rx and SIL were significant for both observers (5.8% to 6.7% v 38.4% to 50.0%, respectively); however, differences between NOS and SIL (30.7% to 42.8% v 38.4% to 50.0%) were not, even when cases were limited to those in which both observers agreed (28.6% v 37.5%). By light microscopy, AISPs exceeding the threshold for presumed reactive changes (NOS or SIL) are a morphologically heterogeneous group that defy precise classification. Furthermore, their histopathologic appearance, even when there is diagnostic agreement, does not consistently correlate with their HPV status. The laboratory management of AISPs should take into account the uncertainty of this diagnosis.

Pseudoinvasion of vascular spaces: report of an artifact caused by cervical lidocaine injection prior to loop diathermy.

Cervical loop diathermy is a relatively new procedure performed by gynecologists to diagnose and treat squamous intraepithelial lesions. We report a case of pseudoinvasion of vascular spaces noted in an excisional biopsy of a high-grade squamous intraepithelial lesion of the cervix. The neoplastic epithelium was forced into the cervical stroma by injection of local anesthetic through the lesion prior to loop diathermy. The identification of this pseudovascular space invasion as artifact had important prognostic and therapeutic value. With the increasing use of loop diathermy and local anesthesia this type of artifact may be seen more commonly.