Complement component 4A protein levels are negatively related to frontal volumes in patients with schizophrenia spectrum disorders.

BACKGROUND: Excessive C4A-gene expression may result in increased microglia-mediated synaptic pruning. As C4A overexpression is observed in schizophrenia spectrum disorders (SSD), this mechanism may account for the altered brain morphology (i.e. reduced volume and cortical thickness) and cognitive symptoms that characterize SSD. Therefore, this study investigates the association of C4A serum protein levels with brain morphology and cognition, and in particular whether this association differs between recent-onset SSD (n = 69) and HC (n = 40). METHODS: Serum C4A protein levels were compared between groups. Main outcomes included total gray matter volume, mean cortical thickness and cognitive performance. Regression analysis on these outcomes included C4A level, group (SSD vs. HC), and C4A*Group interactions. All statistical tests were corrected for age, sex, BMI, and antipsychotic medication dose. Follow-up analyses were performed on separate brain regions and scores on cognitive sub-tasks. RESULTS: The group difference in C4A levels was not statistically significant (p = 0.86). The main outcomes did not show a significant interaction effect (p > 0.13) or a C4A main effect (p > 0.27). Follow-up analyses revealed significant interaction effects for the left medial orbitofrontal and left frontal pole volumes (p < 0.001): C4A was negatively related to these volumes in SSD, but positively in HC. CONCLUSION: This study demonstrated that C4A was negatively related to - specifically - frontal brain volumes in SSD, but this relation was inverse for HC. The results support the hypothesis of complement-mediated brain volume reduction in SSD. The results also suggest that C4A has a differential association with brain morphology in SSD compared to HC.

Proton-electron mass ratio from laser spectroscopy of HD+ at the part-per-trillion level.

Recent mass measurements of light atomic nuclei in Penning traps have indicated possible inconsistencies in closely related physical constants such as the proton-electron and deuteron-proton mass ratios. These quantities also influence the predicted vibrational spectrum of the deuterated molecular hydrogen ion (HD+) in its electronic ground state. We used Doppler-free two-photon laser spectroscopy to measure the frequency of the v = 0→9 overtone transition (v, vibrational quantum number) of this spectrum with an uncertainty of 2.9 parts per trillion. By leveraging high-precision ab initio calculations, we converted our measurement to tight constraints on the proton-electron and deuteron-proton mass ratios, consistent with the most recent Penning trap determinations of these quantities. This results in a precision of 21 parts per trillion for the value of the proton-electron mass ratio.

Intestinal-specific activatable Myb initiates colon tumorigenesis in mice.

Transcription factor Myb is overexpressed in most colorectal cancers (CRC). Patients with CRC expressing the highest Myb are more likely to relapse. We previously showed that mono-allelic loss of Myb in an Adenomatous polyposis coli (APC)-driven CRC mouse model (Apc(Min/+)) significantly improves survival. Here we directly investigated the association of Myb with poor prognosis and how Myb co-operates with tumor suppressor genes (TSGs) (Apc) and cell cycle regulator, p27. Here we generated the first intestinal-specific, inducible transgenic model; a MybER transgene encoding a tamoxifen-inducible fusion protein between Myb and the estrogen receptor-α ligand-binding domain driven by the intestinal-specific promoter, Gpa33. This was to mimic human CRC with constitutive Myb activity in a highly tractable mouse model. We confirmed that the transgene was faithfully expressed and inducible in intestinal stem cells (ISCs) before embarking on carcinogenesis studies. Activation of the MybER did not change colon homeostasis unless one p27 allele was lost. We then established that MybER activation during CRC initiation using a pro-carcinogen treatment, azoxymethane (AOM), augmented most measured aspects of ISC gene expression and function and accelerated tumorigenesis in mice. CRC-associated symptoms of patients including intestinal bleeding and anaemia were faithfully mimicked in AOM-treated MybER transgenic mice and implicated hypoxia and vessel leakage identifying an additional pathogenic role for Myb. Collectively, the results suggest that Myb expands the ISC pool within which CRC is initiated while co-operating with TSG loss. Myb further exacerbates CRC pathology partly explaining why high MYB is a predictor of worse patient outcome.

Different surgical approaches for stress urinary incontinence in women.

Stress urinary incontinence (SUI) constitutes involuntary voiding as a consequence of rising intra-abdominal pressure caused by sphincter weakness. In recent years studies were published according to surgical SUI management evaluating and comparing therapy options and outcomes. Therapy options were evaluated using a Medline search, including only publications in English between 2000-2012. Key words used were: SUI, conservative and surgical treatment, midurethral sling, colposuspension. Surgical treatment options demonstrate significantly better results than conservative treatment. MUS demonstrate better subjective and objective cure rates than colposuspension; it is less invasive and more cost-effective. First line SUI therapy such as RP MUS and TVT seem to be favored when compared to transobturator techniques. Retropubic and transobturator MUS showed equivalent objective and subjective success rates. Open colposuspension is an effective treatment possibility for recurrent SUI after failed MUS. TVT, compared with other MUS, seems to show slightly better cure rates. but perioperative complications appear to be similar. Long-term results (>10 years) of repeated SUI surgery showed that the Burch procedure had the lowest 9-year cumulative incidence of repeat SUI surgery. Mini-sling techniques may be underestimated but long-time results are pending and closer monitoring of the adverse event profile must be carried out. MUS are first choice in the treatment of SUI, of which TVT, has the best cure rate. Colpussupension continues to have its place in recurrent SUI. The new mini-MUS needs a longer follow-up for final evaluation.

[Post-chemotherapy laparoscopic retroperitoneal lymph node dissection in low volume residual germ cell cancer: a technique to reduce morbidity]. / Laparoskopisch retroperitoneale Lymphadenektomie bei Low-volume-Keimzelltumoren nach Chemotherapie : Ein Verfahren zur Verringerung der Morbidität.

PURPOSE: Retroperitoneal lymph node dissection (RPLND) is the most appropriate method for the detection of residual tumor tissue and mature teratoma after chemotherapy in patients with advanced nonseminomatous (NSGCT) or seminomatous (SGCT) germ cell tumors in clinical stage II-III. Open surgical procedures are associated with higher morbidity rates and laparoscopic RPLND offers a minimally invasive procedure with equivalent oncological safety and low morbidity. METHODS: In 39 patients laparoscopic RPLND (L-RPLND) after platinum-based chemotherapy for clinical stage IIa-III NSGCT was performed unilaterally as well as bilaterally by two surgeons. Patients with retroperitoneal residual tumor >1 cm and normalization of tumor markers after chemotherapy were included. Bilateral L-RPLND was performed with complete contralateral nerve sparing while the decision for ipsilateral nerve preservation was based on the volume of the residual mass in the respective standard field. RESULTS: The L-RPLND was completed in all patients without conversion. Median operation time was 248 min (range 95-397 min) and mean hospitalization time was 5 days (range 3-14 days). Furthermore, there was no difference in recurrence rate of the disease (p=0.45) between patients with unilateral or bilateral dissection. The postoperative ejaculatory function was normal in 37 out of 39 patients. The median follow-up period was 18.5 months (range 3-38 months) and 3 out of 39 patients developed recurrence (7.69 %). CONCLUSIONS: Post-chemotherapy L-RPLND is feasible with a lower complication rate and an adequate oncological safety and functional outcome. Due to the complexity of L-RPLND the procedure remains limited to institutions with extensive laparoscopic experience.

[Laparoendoscopic single-site surgery in urology using reusable systems]. / Laparoendoskopische Single-site-Chirurgie in der Urologie durch Einsatz eines wieder verwendbaren Systems.

PURPOSE: Many disposable platforms have been available for laparoendoscopic single-site surgery (LESS) for a long time. Besides technical challenges cost remains the limiting factor for the widespread use of LESS. We present our experiences with the first completely reusable LESS platform. METHODS: We performed LESS procedures in 52 patients, including nephrectomy (n=18), adrenalectomy (2), partial nephrectomy (3), pyeloplasty (4), renal cyst ablation (4), pelvic lymphadenectomy (15) and lymphocele ablation (6). All procedures were carried out using a novel reusable single-port device (X-ConeR, Karl-Storz) with a simplified combination of standard and preformed instruments. Perioperative and demographic data including a visual analogue pain scale (VAS) were obtained. Complications were recorded using the Clavien classification. RESULTS: The mean age of the patients was 50.04 years. Conversion to standard laparoscopy was necessary in 3 cases and the additional use of a 3 mm needle instrument in 6 cases. There were no open conversions. Intraoperative and postoperative complications occurred in 3 (Clavien II in 2 and III in 1) cases. Mean operating time was 110, 90, and 89 min and hospital stay was 4.9, 3.1 and 3.6 days for nephrectomy, pelvic lymphadenectomy, and pyeloplasty, respectively. The mean VAS was 2.13, 1.07 and 1.5 while blood loss was 81.3 ml, 25.67 ml and 17.5 ml, respectively. CONCLUSIONS: The LESS technique with a completely reusable platform is applicable to various indications in urology yielding favorable functional and cosmetic results. This novel simplified combination of instruments facilitates handling and shortens the learning curve. Reusable materials may help to reduce cost leading to a wider acceptance of LESS.

[Minimally invasive percutaneous treatment of lower pole stones with a diameter of 8 to 15 millimeters]. / Die minimalinvasive perkutane Behandlung kleiner unterer Kelchsteine mit einem Durchmesser von 8 bis 15 Millimeter.

PURPOSE: The optimal treatment options for lower pole stones with a diameter below 15 mm are controversially discussed. Extracorporeal shock wave lithotripsy (ESWL) is non-invasive but is hampered by low stone-free rates and a significant retreatment rate. Flexible ureterorenoscopy (URS) has been demonstrated to have high stone-free rates but the treatment costs - consisting of OR time, repair costs and expenditure for laser fibers, guide wires and stone baskets - as well as low stone-free rates with increasing stone size render this procedure highly expensive. Minimally invasive percutaneous litholapaxy (MIP) has shown low morbidity and high efficacy in the treatment of nephrolithiasis. The goal of this study was to investigate the efficacy and -safety of MIP for the treatment of small lower pole stones. PATIENTS AND METHOD: The charts of 29 patients who were treated with MIP were reviewed and clinical data like OR time, drop in haemoglobin, complication rate, stone-free rate and duration of hospital stay were collected. RESULTS: 28 of 29 patients were primarily stone-free; one had to undergo additional flexible URS to become stone-free. All procedures were undertaken with only one access, no severe complications occurred; none of the patients had to be transfused. CONCLUSIONS: The MIP concept has a low complication rate and has been shown to be safe and effective in previous studies. We demonstrate that the feasibility and efficacy justify the percutaneous approach also for small lower pole stones.

Solution structures of casein peptides: NMR, FTIR, CD, and molecular modeling studies of alphas1-casein, 1-23.

To determine its potential for interacting with other components of the casein micelle, the N-terminal section of bovine alphas1-casein-B, residues 1-23, was investigated with nuclear magnetic resonance (NMR), Fourier transform infrared (FTIR) and circular dichroism (CD) spectroscopies, and molecular modeling. NMR data were not consistent with conventional alpha-helical or beta-sheet structures, but changes in N-H proton chemical shifts suggested thermostable structures. Both CD and FTIR predicted a range of secondary structures for the peptide (30-40% turns, 25-30% extended) that were highly stable from 5 degrees C to 25 degrees C. Other conformational elements, such as loops and polyproline II helix, were indicated by FTIR only. Molecular dynamics simulation of the peptide predicted 32% turns and 27% extended, in agreement with FTIR and CD predictions and consistent with NMR data. This information is interpreted in accord with recent spectroscopic evidence regarding the nature of unordered conformations, leading to a possible role of alphas1-casein (1-23) in facilitating casein-casein interactions.

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.

The effect of the erg26-1 mutation on the regulation of lipid metabolism in Saccharomyces cerevisiae.

A temperature-sensitive Saccharomyces cerevisiae mutant harboring a lesion in the ERG26 gene has been isolated. ERG26 encodes 4alpha-carboxysterol-C3 dehydrogenase, one of three enzymatic activities required for the conversion of 4,4-dimethylzymosterol to zymosterol. Gas chromatography/mass spectrometry analyses of sterols in this mutant, designated erg26-1, revealed the aberrant accumulation of a 4-methyl-4-carboxy zymosterol intermediate, as well as a novel 4-carboxysterol. Neutral lipid radiolabeling studies showed that erg26-1 cells also harbored defects in the rate of biosynthesis and steady-state levels of mono-, di-, and triglycerides. Phospholipid radiolabeling studies showed defects in the rate of biosynthesis of both phosphatidic acid and phosphatidylinositol. Biochemical studies revealed that microsomes isolated from erg26-1 cells contained greatly reduced 4alpha-carboxysterol-C3 dehydrogenase activity when compared with microsomes from wild type cells. Previous studies have shown that loss of function mutations in either of the fatty acid elongase genes SUR4/ELO3 or FEN1/GNS1/ELO2 can "bypass" the essentiality of certain ERG genes (Ladeveze, V., Marcireau, C., Delourme, D., and Karst, F. (1993) Lipids 28, 907-912; Silve, S., Leplatois, P., Josse, A., Dupuy, P. H., Lanau, C., Kaghad, M., Dhers, C., Picard, C., Rahier, A., Taton, M., Le Fur, G., Caput, D., Ferrara, P., and Loison, G. (1996) Mol. Cell. Biol. 16, 2719-2727). Studies presented here have shown that this sphingolipid-dependent "bypass" mechanism did not suppress the essential requirement for zymosterol biosynthesis. However, studies aimed at understanding the underlying physiology behind the temperature-sensitive growth defect of erg26-1 cells showed that the addition of several antifungal compounds to the growth media of erg26-1 cells could suppress the temperature-sensitive growth defect. Fluorescence microscopic analysis showed that GFP-Erg26p and GFP-Erg27p fusion proteins were localized to the endoplasmic reticulum. Two-hybrid analysis indicated that Erg25p, Erg26p, and Erg27p, which are required for the biosynthesis of zymosterol, form a complex within the cell.

Validity of lower extremity strength and power utilizing a new closed chain dynamometer.

PURPOSE: The purpose of this study was to compare selected variables measured on a traditional isokinetic dynamometer (Cybex II) with a new lower extremity, closed chain dynamometer (Omnikinetic, OmK). METHODS: Twelve subjects (6 male, 6 female, age = 28+/-5 yr, mean +/- SD) performed Cybex II knee flexion and extension at 1.05, 3.14, and 5.23 rad x s(-1). A maximal effort of 10 repetitions of lower extremity concentric extension and eccentric flexion at 36% of subject's 1-RM was performed on the OmK. Crank power and joint (ankle, knee, and hip) kinetics were recorded as a mean of 10 repetitions. RESULTS: t-Tests revealed right versus left leg differences (P < 0.05) for Cybex II peak torque flexion at 5.23 rad x s(-1), and OmK knee and hip peak power and hip root mean square power (RMS) power. Cybex peak knee torques were related (Pearson r values 0.78-0.92, P < 0.01) to OmK peak knee torques. Cybex average power was related to OmK knee power (Pearson r values 0.71-0.96, P < 0.01) and OmK crank power (r = 0.62-0.94, P < .01). Correlations tended to be stronger comparing the OmK with the fastest (5.23 rad x s(-1)) Cybex II speed. CONCLUSIONS: These results suggest that the OmK knee and crank kinetic data are comparable to Cybex It isokinetic dynamometry. The ability to evaluate lower extremity joint exercise at a subject's maximal movement speed, in addition to the use of a closed-chain, multi-joint motion, may allow for the OmK to provide a more global evaluation of lower extremity kinetics during seated concentric-extension, eccentric-flexion exercise.

Purification and characterization of recombinant forms of murine Tcl1 proteins.

The TCL1 gene, which is located on chromosome 14, plays a major role in human hematopoietic malignancies and encodes a 14-kDa protein whose function has not been determined. This gene is expressed in pre-B cells, in immature thymocytes, and, at low levels, in activated T cells but not in peripheral mature B cells and in normal cells. The Tcl1 protein is similar in its primary structure to a protein encoded by the mature T-cell proliferation gene (MTCP1). The MTCP1 gene is located on the X chromosome and has been shown to be involved in rare chromosomal translocations in T-cell proliferative diseases. The murine TCL1 gene resides on mouse chromosome 12 and is homologous to the human TCL1 and MTCP1 genes. Murine Tcl1 protein has 116 amino acid residues and shares 50% sequence identity with human Tcl1, while the human and mouse Mtcp1 are nearly identical, with conservative differences in only six residues. The TCL1 and MTCP1 genes appear to be members of a family of genes involved in lymphoid proliferation and T-cell malignancies. Our laboratory has undertaken the study of the Tcl1 and Mtcp1 proteins to determine the structure and the function of these related proteins. In the present report, we have produced, using a bacterial expression system, the purified murine Tcl1 protein and a mutant form of murine Tcl1 protein containing a cysteine to alanine mutation at amino acid position 85. The recombinant proteins were purified by chromatography on a Ni-NTA resin followed by reverse-phase FPLC using a buffer system at pH 7.9 and a polymer-based reverse-phase column. The murine Tcl1 recombinant protein displays limited solubility and forms disulfide-linked dimers and oligomers, while the mutant murine Tcl1 C86A protein has increased solubility and does not form higher order oligomers. The purified recombinant murine proteins were characterized by N-terminal sequence analysis, mass spectrometry, and circular dichroism spectroscopy. Initial results indicate that the mutant murine Tcl1 C86A protein is suitable for both NMR and X-ray crystallographic methods of structure determination.

Design of peptides with high affinities for heparin and endothelial cell proteoglycans.

Proteoglycan-binding peptides were designed based on consensus sequences in heparin-binding proteins: XBBXBX and XBBBXXBX, where X and B are hydropathic and basic residues, respectively. Initial peptide constructs included (AKKARA)(n) and (ARKKAAKA)(n) (n = 1-6). Affinity coelectrophoresis revealed that low M(r) peptides (600-1,300) had no affinities for low M(r) heparin, but higher M(r) peptides (2,000-3,500) exhibited significant affinities (K(d) congruent with 50-150 nM), which increased with peptide M(r). Affinity was strongest when sequence arrays were contiguous and alanines and arginines occupied hydropathic and basic positions, but inclusion of prolines was disruptive. A peptide including a single consensus sequence of the serglycin proteoglycan core protein bound heparin strongly (K(d) congruent with 200 nM), likely owing to dimerization through cysteine-cysteine linkages. Circular dichroism showed that high affinity heparin-binding peptides converted from a charged coil to an alpha-helix upon heparin addition, whereas weak heparin-binding peptides did not. Higher M(r) peptides exhibited high affinities for total endothelial cell proteoglycans (K(d) congruent with 300 nM), and approximately 4-fold weaker affinities for their free glycosaminoglycan chains. Thus, peptides including concatamers of heparin-binding consensus sequences may exhibit strong affinities for heparin and proteoglycans. Such peptides may be applicable in promoting cell-substratum adhesion or in the design of drugs targeted to proteoglycan-containing cell surfaces and extracellular matrices.

Conformational dynamics in mixed alpha/beta-oligonucleotides containing polarity reversals: a molecular dynamics study using time-averaged restraints.

Nucleic acid duplexes featuring a single alpha-anomeric thymidine inserted into each DNA strand via 3'-3' and 5'-5' phosphodiester linkages exhibit local conformational dynamics that are not adequately depicted by conventional restrained molecular dynamics (rMD) methods. We have used molecular dynamics with time-averaged NMR restraints (MDtar) to explore its applicability to describing the conformational dynamics of two alpha-containing duplexes--d(GCGAAT-3'-3'-alphaT-5'-5'-CGC)2 and d(ATGG-3'-3'-alphaT-5'-5'-GCTC) x r(gagcaccau). In contrast to rMD, enforcing NOE-based distance restraints over a period of time in MDtar rather than instantaneously results in better agreement with the experimental NOE and J-data. This conclusion is based on the dramatic decreases in average distance and coupling constant violations (delta d(av), J(rms), and delta J(av)) and improvements in sixth-root R-factors (R(X)). In both duplexes, the deoxyribose ring puckering behavior predicted independently by pseudorotation analysis is portrayed remarkably well using this approach compared to rMD. This indicates that the local dynamic behavior is encoded within the NOE data, although this is not obvious from the local R(X) values. In both systems, the backbone torsion angles comprising the 3'-3' linkage as well as the (high S-) sugars of the alpha-nucleotide and preceding residue (alpha - 1) are relatively static, while the conformations of the 5'-5' linkage and the sugar in the neighboring beta-nucleotide (alpha + 1) show enhanced flexibility. To reduce the large ensembles generated by MDtar to more manageable clusters we utilized the PDQPRO program. The resulting PDQPRO clusters (in both cases, 13 structures and associated probabilities extracted from a pool of 300 structures) adequately represent the structural and dynamic characteristics predicted by the experimental data.

Solution structure of a DNA.RNA hybrid containing an alpha-anomeric thymidine and polarity reversals: d(ATGG-3'-3'-alphaT-5'-5'-GCTC). r(gagcaccau).

We report the thermodynamic and structural properties of an alpha-containing DNA.RNA nonamer hybrid duplex, d(ATGG-3'-3'-alphaT-5'-5'-GCTC).r(gagcaccau). The RNA strand corresponds to the core of the initiation sequence for the transcript of the erbB-2 oncogene. The tandem anomeric and polarity changes in the DNA strand result in a slight decrease in thermostability (DeltaT(m) = -2.8 degrees C) compared to the unmodified control hybrid. The three-dimensional solution structure determination of the alpha-containing DNA.RNA hybrid, conducted via restrained molecular dynamics using interproton distance (nuclear Overhauser enhancement) and furanose ring torsion angle (J-based) restraints, converged to a final ensemble of structures from unique starting models. In agreement with hyperchromicity and circular dichroism data, the final average structure derived from this ensemble is consistent with an overall A-like motif featuring Watson-Crick base pairing and base stacking across the entire sequence, albeit with localized B-like traits within the DNA strand. Comparative pseudorotation analyses of the J-coupling data for this hybrid and its unmodified control reveal a surprising increase in S-puckering for two nucleotides immediately upstream of the 3'-3' linkage, and the associated narrowing of the minor groove in this portion of the hybrid. Other structural perturbations are localized to and diagnostic of the central alpha-nucleotide and juxtaposed polarity reversals. The structural information presented here has direct relevance to the design of future antisense oligonucleotides composed of these modifications.

Conformational analysis of the hydrophobic peptide alphas1-casein(136-196).

Hydrophobic interactions are important in the self-association of milk proteins, including alphas1-casein. The extent to which casein interaction sites are influenced by local secondary structure is not widely known. Both primary amino acid sequence and local secondary structure are shown to affect the self-association of the hydrophobic peptide alphas1-casein(136-196). The peptide is aggregated at low concentrations (7 microM and above), as determined by 1H nuclear magnetic resonance (NMR) measurements at pH 6.0 in phosphate buffer. Increase in temperature is shown to induce side chain mobility (melting) as indicated by both 1H NMR and near-UV circular dichroism (CD) measurements. As determined by far-UV CD, there is also a loss in the global amount of extended structure with increasing temperature, while beta-turn structures and some aromatic dichroism are conserved at temperatures as high as 70 degrees C. Similar retention of structure occurs at pH 2 and in 6 M guanidine HCl. The observed stability of beta-turns and some side chains in alphas1-casein(136-196) supports previous assumptions that hydrophobic, proline-based turns are important interaction sites in the self-association of alphas1-casein, and possibly in the formation of the calcium transport complexes, the casein micelles. It may be speculated that these areas of the peptide represent a 'molten globule-like', heat stable, core structure for alphas1-casein.

NMR solution structures of [d(GCGAAT-3'-3'-alphaT-5'-5'-CGC)2] and its unmodified control.

We present the high-resolution solution structures of a self-complementary DNA decamer duplex featuring a single alpha-anomeric nucleotide per strand encompassed by a set of 3'-3' and 5'-5' phosphodiester linkages, d(GCGAAT-3'-3'-alphaT-5'-5'-CGC)2, alphaT, and its unmodified control, d(GCGAATTCGC)2, obtained by restrained molecular dynamics. Interproton distance and deoxyribose ring torsion angle restraints were deduced from homonuclear NOESY and DQF-COSY data, respectively. For both the control and alphaT duplexes, excellent global convergence was observed from two different (A- and B-) starting models. The final average structures of the two duplexes are highly homologous, and overall possess the traits characteristic of right-handed B-DNA duplexes. However, localized differences between the two structures stem from the enhanced conformational exchange in the deoxyribose ring of the cytidine following the 5'-5' linkage, the C3'- exo pseudorotation phase angle of the alpha-nucleotide, and unusual backbone torsions in the 3'-3' and 5'-5' phosphodiester linkages. The structural data reported here are relevant to the design of antisense therapeutics comprised of these modifications.

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.