Burkitt Lymphoma Mimicking an Acute Appendicitis in a 17 Years Old Boy: a Case Report.

Burkitt lymphoma belongs to the B cell non-Hodgkin tumors and is known as the fastest growing human tumor. It is mostly seen in children and young adults. Typically for this type of lymphoma is the chromosomal translocation that leads to a deregulated expression of the c-myc oncogene. Our case report describes a 17 year old patient who presents with atypical right lower quadrant pain. He underwent an explorative laparoscopy and a Burkitt lymphoma was discovered. Subsequently several imaging studies were performed to stage the disease. Intensive systemic chemotherapy is the choice of treatment. Surgery can have a role in early stages and acute complications of the tumor but frequently the role of surgery is restricted to histological biopsy.

Alveolar echinococcosis in a Belgian urban dweller.

Human alveolar echinococcosis is a rare parasitic disease caused by larvae of the tapeworm E. multilocularis that colonizes the intestines of foxes. The disease predominantly affects the liver and mimics slow growing liver cancer. With a mere 13 reports coming mostly from southern rural regions Belgium has so far been spared from the disease. However alveolar echinococcosis appears to be slowly spreading to non-endemic European countries like Belgium and to urban centres. We report the first autochthonous case involving a patient having lived exclusively in downtown Brussels. Heightened awareness by the medical community is necessary to detect this lethal disease at an early curable stage. In patients with an undetermined focal liver lesion--especially if calcified--and no firm evidence of malignancy, serological screening should be performed to exclude alveolar echinococcosis.

Intercalation of daunomycin into d(CG)4 oligomer duplex containing G x T mismatches by vibrational circular dichroism and infrared absorption spectroscopy.

The vibrational circular dichroism (VCD) and infrared absorption (IR) spectra of the mismatched octamer oligonucleotides d(CGTGCGCG)(2) (CGT) and d(CGCGTGCG)(2) (CGC) and their complexes with the antitumor drug daunomycin were measured in D(2)O, interpreted, and compared to the octamer d(CGCGCGCG)(2) (CG). The IR spectra of the mismatched octamers in the carbonyl-stretching region are similar to those of the parent CG, whereas the VCD spectra differ in several respects between each other. The main VCD feature due to carbonyl stretching is informative for the mismatches and CG. Vibrational modes in the sugar-phosphate region remain essentially unchanged especially for PO(2) (-) symmetric stretching. Differences between the free and complexed mismatch octamers occurred mainly in the carbonyl-stretching region (1,700-1,600 cm(-1)). The absorption intensity of the C==O peak of G is more prominent for CGC than CGT and resembles CG in this respect. The detailed composition of this doublet is clearly visible, indicating the geometric rearrangement of the base pairs in the presence of the mismatch and upon forming the daunomycin complex.

Vibrational circular dichroism signature of hemiprotonated intercalated four-stranded i-DNA.

The four-stranded intercalated DNA structure exemplified by the oligonucleotide 5'-d(CCCCCCCCCCCC) (d(C)12) was studied at acidic pH by infrared absorption (IR) and vibrational circular dichroism (VCD) spectroscopy and compared with spectra of the same oligonucleotide at neutral pH to establish distinct VCD markers for the intercalation motif. The most striking feature is a new absorption at 1694 cm(-1) and its corresponding VCD couplet with reversed sign. These are unique for the intercalated structure and have not been observed for other parallel stranded duplexes. Significant characteristic features resulting from the spatial arrangement of the sugar-phosphate backbone are also clearly present for d(C)12 at acidic pH. An extensive network of CH...O bonds twists the backbone such that multiple through-space vibrational coupling occurs among neighbouring sugar-phosphate residues resulting in unusual VCD signals.

Cisplatin adducts of d(CCTCTG*G*TCTCC).d(GGAGACCAGAGG) in aqueous solution by vibrational circular dichroism spectroscopy.

The vibrational circular dichroism (VCD) and IR absorption spectra of a dodecamer d(CCTCTGGTCTCC).d(GGAGACCAGAGG) coordinated with cisplatin are distinct compared to those of the dodecamer without cisplatin. Although the intensity of PO(2)/deoxyribose absorptions (1150-850 cm(-1)) increases noticeably relative to those of the carbonyl and ring deformations of the bases (1750-1500 cm(-1)), the VCD spectra differ to a much greater extent. Overlapping positive and negative bands can be assigned relatively easily to individual vibrational modes. The effect of platination on the dodecamer duplex is expressed most prominently in VCD arising solely from the vibrations of the guanines bound to the platinum atom. The effect on the VCD features of other bases leads to minute wavenumber shifts at most. These observations are in agreement with previous NMR and X-ray experiments on the same oligonucleotide. The assignment of the absorption and VCD bands strongly resembles those of the octamer duplex d(CCTGGTCC).d(GGACCAGG) when coordinated with platinum. The spectra of the dodecamer did not indicate any isomerization of the complex with time, as is clearly the case for the octamer.

Vibrational circular dichroism and IR absorption of DNA complexes with Cu2+ ions.

Vibrational circular dichroism (VCD) spectroscopy and simultaneous IR absorption measurements are applied to study the interaction of natural calf thymus DNA with Cu2+ ions at room temperature in a Cu2+ concentration range of 0-0.4M (a Cu2+/phosphate molar ratio [Cu]/[P] of 0-0.7). In some important instances, VCD provides more detailed insights than previous IR investigations whereas in several others it leads to the same interpretations. The Cu2+ ions bind to phosphate groups at a low metal concentration. Upon increasing the ion concentration, chelates are formed in which Cu2+ binds to the N7 of guanine (G) and a phosphate group. Detectable only by VCD, significant distortion of most guanine-cytosine (GC) base pairs occurs at a [Cu]/[P] ratio of 0.5 with only a minor affect on adenine-thymine (AT) base pairs, which favors a "sandwich" complex in which a Cu2+ ion is inserted between two adjacent guanines in a GpG sequence. The AT base pairs become significantly distorted when the metal concentration is increased to 0.7 [Cu]/[P]. A number of GC base pairs, which are possibly involved in sandwich complexes, remain stacked and paired even at 0.7 [Cu]/[P], preventing complete strand separation. The DNA secondary structure changes considerably from the standard B-form geometry at a [Cu]/[P] ratio of 0.4 and higher. A further transition to some intermediate conformation that is inconsistent with either the A- or Z-form or a completely denatured state is suggested in agreement with other works. In general, VCD proves to be a reliable indicator of the 3-dimensional structure of the DNA-metal ion complexes, which reveals structural details that cannot be deduced from the IR absorption spectra alone.

Poly(rA).poly(rU) with Ni(2+) ions at different temperatures: infrared absorption and vibrational circular dichroism spectroscopy.

Phase transitions were studied of the sodium salt of poly(rA).poly(rU) induced by elevated temperature without Ni(2+) and with Ni(2+) in 0.07 M concentration in D(2)O (approximately 0.4 [Ni]/[P]). The temperature was varied from 20 degrees C to 90 degrees C. The double-stranded conformation of poly(rA).poly(rU) was observed at room temperature (20 degrees C-23 degrees C) with and without Ni(2+) ions. In the absence of Ni(2+) ions, partial double- to triple-strand transition of poly(rA).poly(rU) occurred at 58 degrees C, whereas only single- stranded molecules existed at 70 degrees C. While poly(rU) did not display significant helical structure, poly(rA) still maintained some helicity at this temperature. Ni(2+) ions significantly stabilized the triple-helical structure. The temperature range of the stable triple-helix was between 45 degrees C and 70 degrees C with maximum stability around 53 degrees C. Triple- to single-stranded transition of poly(rA).poly(rU) occurred around 72 degrees C with loss of base stacking in single-stranded molecules. Stacked or aggregated structures of poly(rA) formed around 86 degrees C. Hysteresis took place in the presence of Ni(2+) during the reverse transition from the triple-stranded to the double-stranded form upon cooling. Reverse Hoogsteen type of hydrogen-bonding of the third strand in the triplex was suggested to be the most probable model for the triple-helical structure. VCD spectroscopy demonstrated significant advantages over infrared absorption or the related electronic CD spectroscopy.

Structural, dynamic, and enzymatic properties of mixed alpha/beta-oligonucleotides containing polarity reversals.

We employ NMR structure determination, thermodynamics, and enzymatics to uncover the structural, thermodynamic and enzymatic properties of alpha/beta-ODNs containing 3'-3' and 5'-5' linkages. RNase H studies show that alpha/beta-gapmers that are designed to target erbB-2 efficiently elicit RNase H activity. NMR structures of DNA.DNA and DNA.RNA duplexes reveal that single alpha-anomeric residues fit well into either duplex, but alter the dynamic properties of the backbone and deoxyriboses as well as the topology of the minor groove in the DNA.RNA hybrid.

Complexes of (dG-dC)20 with Mn2+ ions: a study by vibrational circular dichroism and infrared absorption spectroscopy.

The B-Z transition of the synthetic oligonucleotide, (dG-dC)20, induced by Mn2+ ions at room temperature, was investigated by absorption and Vibrational Circular Dichroism (VCD) spectroscopy in the range of 1800-800 cm(-1). Metal ion concentration was varied from 0 to 0.73 M Mn2+ (0 to 8.5 moles of Mn2+ per mole of oligonucleotide phosphate, [Mn]/[P]). While both types of spectra showed considerable changes as the Mn2+ concentrations were raised, differences between the two were often complementary in their expression and extent, those displayed by VCD being more clearly evident due to the inversion of the opposite helical sense from the right-handed to the left-handed conformation. The main phase of the transition occurred in the metal ion concentration between 0.8-1.1 [Mn]/[P]. Gradual changes that took place in the spectra were interpreted in terms of simultaneous processes that depended on metal ion concentration, namely B-Z transformation, binding of Mn2+ to phosphates and to nitrogen bases, and partial denaturation. Below approximately 0.6 [Mn]/[P], only a small portion of the oligonucleotide adopted the Z conformation within a 3 hour period, whereas conversion was completed in the same time interval for concentrations between 0.9-1.2 [Mn]/[P]. At [Mn]/[P] >1.7, complete transition to the Z-form took place immediately on adding Mn2+. Applying VCD spectroscopy in combination with conventional infrared absorption proved most useful for corroborating changes in the absorption spectra, and for detecting in an unique manner, not attainable by absorption methods, conformational changes that lead to the inversion of the helical sense of the oligonucleotide.

Structure of d(GT)n.d(GA)n sequences: formation of parallel stranded duplex DNA.

Alternating polypurine sequences exhibit remarkable polymorphism. In this study, we report that dGA.dGT sequences form parallel stranded duplex DNA at neutral pH. Using two model hairpins, 3'-d(GT)3-5'5'-T4(AG)3-3' (I) and 3'-d(GT)4-5'5'-T4(AG)4-3' (II), containing 5'5' linkages which direct parallel strand formation, we systematically explored the spectroscopic and thermodynamic properties of parallel stranded d(GA)n.d(GT)n. The parallel stranded hairpins are remarkably stable structures with TM's of 41.5 and 47.5 degreesC (in 0.4 M NaCl) for the shorter and longer hairpins, respectively. The van't Hoff enthalpies of 80.7 and 114 kJ mol-1 are relatively low but are comparable to a parallel stranded d(GA)n duplex. On the basis of the spectroscopic and electrophoretic characteristics, we conclude that parallel strand formation is not restricted to hairpin systems, but also readily occurs in unconstrained dimeric duplexes with the appropriate sequence homologies. Both melting curves and electrophoretic analyses of parallel stranded heteroduplexes in which the sequence enforces specific base pairing demonstrate that G-G and A-T base pairs are formed in d(GA)n.d(GT)n segments.

Antiparallel DNA duplex formation between alternating alpha d(GA)n and beta d(GA)n sequences.

Alternating polypurine d(GA)n, sequences exhibit a considerable polymorphism. Here we report that alpha d(GA) x d(GA) sequences form an antiparallel stranded duplex DNA at neutral pH. The spectroscopic, electrophoretic and thermodynamic properties of the alpha/beta chimeric oligodeoxynucleotide, 5'-d(GA)4(T)4 alpha d(AG)4T-3', support the formation of a hairpin structure with antiparallel strands in the stem. The optical properties of this novel antiparallel structure are different from the parallel stranded homoduplex formed by d(GA)G7. This alpha/beta hairpin has a remarkably high Tm of 44.5 degrees C in 0.4 M NaCl with a van't Hoff enthalpy comparable to that of a parallel d(GA)n duplex. Base pairing was confirmed by T4 polynucleotide ligase catalyzed joining of the alpha/beta hairpin to an antiparallel bimolecular duplex and by non-denaturing gel electrophoresis using duplexes containing sequence constraints. Both support the presence of alphaG-G and alphaA-A base pairing in the antiparallel 5'-d(GA)4(T)4 alpha d(AG)4T-3' intramolecular duplex. This study adds to the polymorphic nature of alternating d(GA)n sequences as well as providing novel homopurine base pairing approaches for probing polypurine polypyrimidine sequences.

Biologic activity of oligonucleotides with polarity and anomeric center reversal.

Human papillomavirus (HPV) type 16 E6 and E7 inactivate the tumor suppressors p53 and pRB, respectively. Both viral oncoproteins play important roles in maintaining the transformed phenotype of cells. In this study, we examine the effects of antisense oligodeoxynucleotides with polarity and anomeric center reversal (alpha/beta-ODNs). ODNs of the general structure 5'alphaN3'3'NNN5'5'alphaN3'3'NNNN5'5'alphaN3+ ++'3'N5' were synthesized using phosphoramidite DNA chemistry. These alpha/beta-ODNs were complementary in sequence to regions flanking the start codons of HPV type 16 E6 and E7 genes. The anti-HPV type 16 alpha/beta-ODNs were able to form stable duplexes with their complementary RNA, which then serve as substrates for RNase H hydrolysis. Anti-HPV type 16 alpha/beta-ODNs also specifically inhibited the growth of two cervical carcinoma cell lines, CaSki and SiHa, both of which harbor HPV type 16 DNA. A decrease in E7 protein expression was also observed. Injection of nude mice with SiHa cells induces tumors. Treatment of these tumor-bearing mice with anti-HPV type 16 alpha/beta-ODNs led to substantially smaller tumors. These results show that alpha/beta-ODNs can exert antisense activities both in vitro and in vivo on the E6 and E7 genes of HPV type 16.

NMR spectroscopic and enzymatic studies of DNA hairpins containing mismatches in the EcoRI recognition site.

We have correlated the structural perturbations caused by DNA mismatches with the enzymatic data of the interaction of the restriction endonuclease EcoRI with DNA. Oligonucleotides d(CGAGAATTCTCA5GAXAATTCT) (X = G, A, T) and d(CGCGAATTYGCGT4CGCXAATTCGCG) (Y = C, X = G, T and Y = A, X = T) containing single mismatches within the EcoRI recognition site were characterized by NMR spectroscopy and by their EcoRI substrate properties. UV melting and gel electrophoresis studies confirm that the oligonucleotides form hairpin structures. The presence of either a CT or a CA mismatch results in markedly lower Tm and van't Hoff enthalpies compared with the fully base paired control. NMR imino proton spectra of these hairpins demonstrate that the perturbation caused by the two mispairs or a noncanonical AT pair is localized and limited to one or two base pairs on either side of the perturbation. The DNA hairpin structures containing single mismatches, and to a lesser extent also sequences with a single noncanonical base pair, are substrates for the restriction endonuclease. In addition to the strand scission at the nonperturbed GpA phosphodiester bond some cleavage is observed at the mismatched position. The interactions of the CA and CT mismatched hairpin with the enzyme are characterized by binding constants that are only 33 and 57 times lower, respectively, than that for the canonical sequence, corresponding to 8-10 kJ x mol(-1) less favorable free binding energy. This, taken together with the NMR data, indicates that the CA and CT mismatches have only small effects on the EcoRI recognition of the DNA substrate. We conclude that two out of the three hydrogen bonds that characterize the interaction of EcoRI with the CG base pair in the canonical sequence can still be formed for either the CT or CA mismatched recognition site.

Spectroscopic and thermodynamic studies of DNA duplexes containing alpha-anomeric C, A, and G nucleotides and polarity reversals: coexistence of localized parallel and antiparallel DNA.

We present a thermodynamic, enzymatic, and spectroscopic study of three self-complementary DNA decamer duplexes, d[GCGAATT-3'-3'-(alphaC)-5'-5'-GC]2 (alphaC), d[GCG-3'-3'-(alphaA)-5'-5'-ATTCGC]2 (alphaA), and d[GC-3'-3'-(alphaG)-5'-5'-AATTCGC]2 (alphaG), which are identical in sequence but contain one alpha-anomeric nucleotide per strand in a parallel orientation via 3'-3' and 5'-5' phosphodiester bonds; the results are placed in the context of our recent studies on the other members of this series, namely alphaT, d[GCGAAT-3'-3'-(alphaT)-5'-5'-CGC]2, and the unmodified control [Aramini, J. M., et al. (1996) Biochemistry 35, 9355-9365]. On the basis of UV hyperchromicity and melting profiles as well as 1H and 31P nuclear magnetic resonance (NMR) spectroscopic data, we conclude that all five constructs form stable duplexes, with very comparable structural features that are consistent with an overall right-handed, antiparallel B-DNA motif and Watson-Crick base pairing throughout. However, each of the alpha-containing sequences exhibits unique thermodynamic and structural differences ascribed to the nature (and position) of the alpha-nucleotide. First, the thermostability of these duplexes decreases from the control to alphaC in the following series: control > alphaT approximately alphaA approximately alphaG > alphaC. Second, in each of the four alpha-duplexes, 1H and 31P chemical shift differences compared to those of the control duplex are largely confined to the region encompassing the alpha-nucleotide and unnatural phosphodiester linkages, as well as neighboring nucleotides. Surprisingly, for alphaC, these modifications result in a significant alteration to the backbone conformation at the phosphodiester group directly across from the 3'-3' linkage. Finally, spin-spin (J) coupling data, specifically Sigma1', indicate that the vast majority of the furanose rings in these duplexes display a high propensity for adopting the S pucker. However, in alphaC, alphaA, and alphaT (but not alphaG), the sugar ring conformation in the nucleotide immediately following the 5'-5' linkage is described by an approximately equal distribution between the N and S conformers.

The mouse uracil-DNA glycosylase gene: isolation of cDNA and genomic clones and mapping ung to mouse chromosome 5.

Uracil-DNA glycosylase (UDG) is the enzyme responsible for the first step in the base-excision repair pathway that specifically removes uracil from DNA. Here we report the isolation of the cDNA and genomic clones for the mouse uracil-DNA glycosylase gene (ung) homologous to the major placental uracil-DNA glycosylase gene (UNG) of humans. The complete characterization of the genomic organization of the mouse uracil-DNA glycosylase gene shows that the entire mRNA coding region for the 1.83-kb cDNA of the mouse ung gene is contained in an 8.2-kb SstI genomic fragment which includes six exons and five introns. The cDNA encodes a predicted uracil-DNA glycosylase (UDG) protein of 295 amino acids (33 kDa) that is highly similar to a group of UDGs that have been isolated from a wide variety of organisms. The mouse ung gene has been mapped to mouse chromosome 5 using fluorescence in situ hybridization (FISH).

Structure of a DNA duplex that contains alpha-anomeric nucleotides and 3'-3' and 5'-5' phosphodiester linkages: coexistence of parallel and antiparallel DNA.

We report a comparative spectroscopic study of a novel self-complementary duplex decamer, d(GCGAAT-3'-3'-(alpha T)-5'-5'-CGC)2, in which an alpha-anomeric nucleotide has been inserted into the sequence in a parallel orientation via 3'-3' and 5'-5' phosphodiester bonds, and its unmodified B-DNA analog, d(GCGAATTCGC)2. Plots of the hyperchromicity and circular dichroism of these oligonucleotides are virtually identical, indicating that the overall base stacking and handedness are preserved in the alpha duplex. Thermodynamic parameters extracted from UV melting experiments show that the alpha duplex is only slightly less stable than the control. A near complete set of 1H and 31P nuclear magnetic resonance (NMR) assignments were obtained for both duplexes using classical one- and two-dimensional approaches. Several lines of evidence, in particular, imino 1H, 31P, nuclear Overhauser enhancement, and deoxyribose ring proton spin-spin coupling data, convincingly demonstrate that the overall structural integrity of the alpha and control duplexes are quite comparable, with any perturbations in the former localized to the regions of the construct encompassing the alpha-nucleotide and the unique backbone linkages. Specifically, the alpha duplex exhibits normal Watson-Crick type base pairing, it remains antiparallel except at the inverted nucleotide, all bases are in the anti orientation, and the sugar ring puckering is predominantly "S"-type. However, the J-coupling information for the alpha-nucleotide and the neighboring (3') cytidine are notably different, and reflect a decrease in the amplitude of the sugar pucker in alpha T7, and a significant shift in the conformational equilibrium of the furanose ring in C8 toward the "N"-type pucker. The feasibility of synthesizing oligodeoxynucleotides containing a combination of alpha sugars and short parallel stranded segments, their propensity for forming stable duplexes, and the structural insights into such complexes reported here are of potential importance in the area of antisense therapy.

Homooligomeric dA.dU and dA.dT sequences in parallel and antiparallel strand orientation: consequence of the 5-methyl groups on stability, structure and interaction with the minor groove binding drug Hoechst 33258.

Oligodeoxyribonucleotides containing dA.dU base combinations were shown to form parallel stranded DNA. CD spectra and hyperchromicity profiles provide evidence that the structure is very similar to that of a related parallel stranded dA.dT oligomer. Thermal denaturation studies show that these parallel dA.dU sequences are significantly less stable than their dA.dT analogues in either antiparallel or parallel stranded orientations. The stabilizing effect of the 5-methyl group is similar for parallel and antiparallel sequences. The minor groove binding drug Hoechst 33258 binds with similar affinity to APS dA.dT and APS dA.dU sequences. However, binding to the PS dA.dT hairpin is significantly impaired as a consequence of the different groove dimensions and the presence of thymine methyl groups at the binding site. This results in an 8.6 kJmol-1 reduced free energy of binding for the PS dA.dT sequence. Replacement of the bulky methyl group with a hydrogen (ie. T-->U) results in significantly stronger Hoechst 33258 binding to the parallel dA.dU sequences with a penalty of only 4.1 kJmol-1. Our data demonstrate that although Hoechst 33258 detects the altered groove, it is still able to bind a PS duplex containing dA.dU base pairs with high affinity, despite the large structural differences from its regular binding site in APS DNA.