Molecular and crystal structure of d(CGCGmo4CG): N4-methoxycytosine.guanine base-pairs in Z-DNA.

The base analogue N4-methoxycytosine (mo4C) is ambivalent in its hydrogen-bonding potential, since it forms stable base-pairs with both adenine and guanine in oligomer duplexes. To investigate the base-pair geometry, the structure of d(CGCGmo4CG) has been determined by single-crystal X-ray diffraction techniques. The d(CGCGmo4CG)2 crystallized in a left-handed double helical structure (Z-type). Refinement using 2559 reflections between 10 and 1.7 A converged with a final R = 0.181 (Rw = 0.130) including 68 solvent molecules. The orthorhombic crystals are in the space group P2(1)2(1)2(1), with cell dimensions a = 18.17 A, b = 30.36 A, c = 43.93 A. The mo4C.G base-pair is of the wobble type, with mo4C in the imino form, and the methoxy group in the syn configuration.

Molecular basis for methoxyamine initiated mutagenesis. 1H nuclear magnetic resonance studies of base-modified oligodeoxynucleotides.

In order to reach a more detailed understanding of the mechanism of the mutagenic action of methoxyamine and of N4-methoxycytidine and its 2'-deoxyribo-analogue, the solution structures of the self-complementary octanucleotide, d(CGAATTCG) and its analogues, d(CGAATCCG), d(CGAATMCG) and d(CGAATPCG) (designated 8mer-AT, 8mer-AC, 8mer-AM, and 8mer-AP, respectively), were investigated by 1H nuclear magnetic resonance spectroscopy; M is N4-methoxycytosine (mo4C) and P is an analogue, the bicyclic dihydropyrimido[4,5-c][1,2]oxazin-7-one, in which the N-O bond is held in the anti configuration with respect to N3 of the cytosine ring. Correlated spectroscopy and nuclear Overhauser spectroscopy allowed assignment of the base, anomeric and H2'/H2" protons in 8mers-AT, -AM and -AP, and showed that all three had features consistent with a regular B-DNA duplex structure. Duplex-to-coil transition temperatures were determined to be 52(+/- 2) degrees C (8mer-AT), 51(+/- 2) degrees C (8mer-AP), 32(+/- 2) degrees C (8mer-AM); on the chemical shift timescale, the melting transition was fast for 8mer-AT and 8mer-AP, but slow for 8mer-AM. Imino proton spectra were indicative of Watson-Crick base-pairing in 8mers-AT, -AP and -AM. The 8mer-AP duplex had a structure and melting characteristics virtually identical with those of the 8mer-AT duplex. The preferred syn configuration of the methoxyl group in M had a destabilising effect on the 8mer-AM duplex. At low temperatures, the A.M base-pair was in fast equilibrium between Watson-Crick and wobble configurations, with the methoxyl function anti-oriented, but the melting transition was accompanied by isomerization of the methoxyl group to the syn conformation. This syn-anti isomerization was the rate-determining step in the duplex-to-coil transition. The 8mer-AC oligomer did not form a stable duplex.

Direct observation of two base-pairing modes of a cytosine-thymine analogue with guanine in a DNA Z-form duplex: significance for base analogue mutagenesis.

The pyrimidine nucleobase analogue 6H,8H-3,4-dihydropyrimido[4,5-c]- [1,2]oxazin-7-one (P) is a mimic both of cytosine and thymine, since it can form stable hydrogen-bonded base-pairs with either guanine or adenine. To investigate the geometric properties of pairing with guanine in a DNA double helix, the structure of d(CGCGPG)2 has been determined by single crystal X-ray analysis. The oligonucleotide crystallised as a left-handed Z-DNA duplex in the orthorhombic space group P2(1)2(1)2(1) with cell dimensions a = 18.23 A, b = 30.63 A, c = 43.78 A. Refinement using NUCLSQ with 51 water molecules included in the final model converged at R = 0.179 (Rw = 0.159) for 2798 reflections (F > 2 sigma (F)) in the range 8 A to 1.7 A. Remarkably, the two P.G pairs in the hexamer duplex are different: Watson-Crick and wobble types separately illustrate both cytosine-like and thymine-like behaviour. The result suggests that mutagenesis experiments involving P and other analogues which display pronounced base-pairing ambivalence can be used to examine the structural basis of substrate discrimination by polymerases that is essential to accurate genetic replication.

Molecular basis for methoxyamine-initiated mutagenesis: 1H nuclear magnetic resonance studies of oligonucleotide duplexes containing base-modified cytosine residues.

Methoxyamine, N4-methoxycytidine and its 2'-deoxyribo analogue are transition mutagens. The mechanism by which the latter acts after incorporation into or generation within DNA has been ascribed to the ability of the base analogue to pair effectively with both adenine and guanine. To obtain a detailed understanding of these interactions, the solution structures of the self-complementary octanucleotide d(CGGATCCG) and its analogues d(CGGATTCG), d(CGGATMCG) and d(CGGATPCG) (designated 8mer-GC, -GT, -GM and -GP, respectively) were investigated by 1H nuclear magnetic resonance spectroscopy; M is N4-methoxycytosine (mo4C) and P is an analogue, the bicyclic dihydropyrimido[4,5-c][1,2] oxazin-7-one. A variable temperature study showed the order of stability as 8mer GC > GP > GT > GM. Nuclear Overhauser spectroscopy permitted the assignment of the base, anomeric and H2'/H2" protons in these 8mers. All had spectra consistent with regular B-DNA duplex structures. Imino proton spectra showed that the 8mers GC, GP and GM involved Watson-Crick base-pairing but that the G.P and to a greater extent G.M base-pairs were in slow exchange on the nuclear magnetic resonance time-scale with the wobble configuration. Indeed, the G.M pair showed an additional exchange process interpreted in terms of the presence of syn and anti conformers of the methoxy group in the wobble pair. This accounts for the destabilization of M compared with the P-containing duplex. The observations are compared with those made earlier on the corresponding AT, AP and AM octamers. It is evident that M and P can form stable base-pairs with both A and G with essentially Watson-Crick geometry. This confirms the earlier, although unsubstantiated explanation for the transition mutational propenstty of methoxyamine which, in turn, was based on the fact that methoxycytosine bases have tautomeric constants (KT) much nearer to unity than the normal bases. The same general explanation for hydroxylamine and hydrazine-induced mutations is correspondingly rendered more certain.

Differential effects of polyamine derivative compounds against Leishmania infantum promastigotes and axenic amastigotes.

The natural polyamines are ubiquitous polycationic compounds that play important biological functions in cell growth and differentiation. In the case of protozoan species that are causative agents of important human diseases such as Leishmaniasis, an exogenous supply of polyamines supports parasite proliferation. In the present study, we have investigated the effect of three polyamine derivatives, (namely bis-naphthalimidopropyl putrescine (BNIPPut), spermidine (BNIPSpd) and spermine (BNIPSpm)), on the proliferative stages of Leishmania infantum, the causative agent of visceral leishmaniasis in the Mediterranean basin. A significant reduction of promastigotes and axenic amastigotes growth was observed in the presence of increasing concentrations of the drugs, although the mechanisms leading to the parasite growth arrest seems to be different. Indeed, by using a number of biochemical approaches to analyse the alterations that occurred during early stages of parasite-drug interaction (i.e. membrane phosphatidylserine exposure measured by annexin V binding, DNA fragmentation, deoxynucleotidyltranferase-mediated dUTP end labelin (TUNEL), mitochondrial transmembrane potential loss), we showed that the drugs had the capacity to induce the death of promastigotes by a mechanism that shares many features with metazoan apoptosis. Surprisingly, the amastigotes did not behave in a similar way to promastigotes. The drug inhibitory effect on amastigotes growth and the absence of propidium iodide labelling may suggest that the compounds are acting as cytostatic substances. Although, the mechanisms of action of these compounds have yet to be elucidated, the above data show for the first time that polyamine derivatives may act differentially on the Leishmania parasite stages. Further chemical modifications are needed to make the polyamine derivatives as well as other analogues able to target the amastigote stage of the parasite.

Effect of three novel polyamine oxa-analogues (MTR-OSPD, DIP-SPN and APPO-TFA) on the growth and proliferation of Swiss 3T3 cells.

In order to investigate their biological function on cellular polyamine content, cell growth and proliferation, three novel polyamine oxa-analogues, 5-(4-methoxy-2,3,6-trimethylbenzenesulfonyl)-6-oxa-spermidine (MTR-OSPD); 6,9-dioxa-5,10-di-(2,2,5,7,8-pentamethylchroman-6-sulfonyl) spermine (DIP-SPN) and 3-aminopropyl N-(3-phthalimidopropyloxy) trifluoroacetimidate (APPO-TFA) were tested for their ability to stop or slow down the growth of Swiss 3T3 cells. Cells at 50-60% confluency were grown for 24 or 48 hr in the presence of a wide range of polyamine oxa-analogue concentrations and the number of cells counted. To determine whether the drugs were cytotoxic or cytostatic, the analogue-containing medium in some vials was replaced with fresh culture medium after 48 hr and the cells incubated for a further 24 hr. Cellular protein, RNA, DNA, polyamine contents and the activities of ornithine decarboxylase, S-adenosyl-L-methionine decarboxylase and spermidine/spermine N1-acetyltransferase were also determined at the lowest effective analogue concentration. All three inhibitors stopped cell proliferation at concentrations over 100 microM. Both MTR-OSPD and DIP-SPN were cytotoxic, since the cells could not be revived by removing the inhibitor from the medium, whereas APPO-TFA was only cytostatic. At the lowest effective concentration the analogues had little effect on protein, RNA and DNA content of the cells, but had varying effects on polyamine metabolism. The most interesting analogue was APPO-TFA. This drug showed concentration-dependent growth inhibition between concentrations of 5 nM and 5 microM. These novel analogues may be of value in elucidating the precise functions of polyamines in cellular metabolism. Their exact mode of action is now under investigation.

Repeated pregnancy without lactation: effects on maternal glycemic control, pregnancy outcome, carcass composition, and fat distribution in rats.

Repeated pregnancy without lactation in rats has been reported to produce permanently elevated carcass fat mass and hyperplasia in subcutaneous fat depot. In the present study, the pregnancy outcome, intravenous glucose tolerance ability (IVGTT), and carcass composition were further examined in Osborne-Mendel rats. Female rats were divided into pregnancy-lactation (PL), pregnancy-no lactation (PNL), and control (CON) groups. Half of the rats were killed after three pregnancy/lactation cycles, whereas the rest were given a 12-week rest period before killing. It was found that rats in PNL group had fasting hyperglycemia and insulin insensitivity during the third pregnancy (P less than or equal to .05), elevated spontaneous abortion rate (P less than or equal to .05), and elevated subcutaneous fat content (P less than or equal to .01) relative to that of PL rats or CON rats. After a 12-week rest period, a significant increase in subcutaneous fat cell number was also observed in PNL rats. PL rats, on the other hand, have significantly elevated internal/subcutaneous fat ratios (P less than or equal to .05), both immediately after weaning and after the 12-week rest period. The significance of this change is not yet known.

Estimation of body composition changes during weight cycling by bioelectrical impedance analysis in rats.

The effects of body weight cycling (WC) in rats on body composition (BC) and feeding efficiency were studied. The usefulness of estimating BC by bioelectrical impedance analysis (BIA) was also examined. Female Sprague-Dawley rats were divided into high-fat ad libitum feeding, either noncycling or cycling, or restricted feeding (75% of control feed) cycling groups. Control rats were fed a regular laboratory ad libitum diet and did not cycle. All rats were killed at the end of week 61. A BIA unit was used at each stage of WC to obtain resistance and reactance readings. Final BC was determined by chemical analysis. On the basis of the final chemical analysis and BIA measurements, an equation was established and applied to estimate BC at each stage of WC: fat-free mass (g) = 0.38 x body wt (g) + 13.8 x [length (cm)2/resistance] + 70.9 (r = 0.95, P < 0.001). High-fat ad libitum feeding induced rapid body weight and fat gains as well as an elevated feeding efficiency and an internal fat-to-subcutaneous fat ratio, regardless of whether the rats cycled. This change in fat mass was clearly detected by the BIA. Although rats fed restricted diets had similar body weights as did control rats, they had a significantly higher internal fat-to-subcutaneous fat ratio. Thus, not only the amount of food but also the composition of the diet is important for proper weight management. The BIA method is capable of detecting the body fat mass change during WC.

Noninvasive glucose monitoring with optical heterodyne technique.

A novel optical polarimeter to sense the glucose level in vivo and noninvasively has been developed in the Institute of Radiological Science, National Yang Ming University. A 30-minute delay between the aqueous glucose and the blood glucose was observed in rabbit's eyeball. Currently, aqueous glucose in low concentration is being tested and discussed.

Synthesis and duplex stability of oligonucleotides containing adenine-guanine analogues.

The nucleosides N6-methoxy-2'-deoxyadenosine (dZ) and 2-amino-9-(2-deoxy-beta-ribofuranosyl)-6-methoxyaminopurine (dK) have been synthesised and converted into 5'-O-dimethoxytrityl 3'-(2-cyanoethyl N,N-diisopropylphosphoramidites). These monomers have been used in machine DNA synthesis to give a set of heptadecanucleotides containing up to three analogue nucleotides. The melting transitions (Tm) show that the 17-mer duplexes containing Z.T and Z.C base-pairs have closely similar stabilities, as have those containing K.T and K.C pairs. They are less stable than the corresponding fully complementary duplexes, but more stable than those containing mismatched pairs. This, in the case of dZ, is in accord with the amino-imino tautomeric ratio of approximately 1:4 observed for the nucleoside in methyl sulfoxide. The application of oligomers containing such "degenerate" bases in oligonucleotide probes and primers is discussed.

Partial hydrodynamic screening of confined linear and circular double-stranded DNA dynamics.

We performed experiments and simulations to investigate the influence of hydrodynamic interaction on the diffusion dynamics of circular and linear λ-DNA confined in nanoslits. Contrary to the common assumption that intrachain hydrodynamic interaction (HI) is completely screened when polymers are confined in channels with height h smaller than the radius of gyration R(g), it is found that the HI is partially screened and approaches complete screening only for R(g)≪h. For λ-DNA, the HI becomes nearly completely screened only when the channel height is smaller than the Kuhn length. In addition, the dynamics of linear and circular λ-DNA in very strong confinement is shown to be independent of the chain topology.

Synthesis and duplex stability of oligonucleotides containing cytosine-thymine analogues.

The synthesis of the deoxynucleoside derived from the base P, 6H,8H-3,4-dihydro-pyrimido[4,5-c] [4,5-c] [1,2]oxazin-7-one, 2, and its introduction by established phosphoramidite and H-phosphonate chemistry into oligonucleotides is described. The melting transition temperatures (Tm) of a range of heptadecamer duplexes containing P/A and P/G base-pairs are compared with corresponding ones having N4-methoxycytosine (M) 1 and mismatched normal bases. P/A and P/G pairs allow closely similar duplex stabilities and have the potential to reduce the multiplicity of probes and primers based on amino acid sequences by removing the T/C degeneracy.

The structure and application of oligodeoxyribonucleotides containing modified, degenerate bases.

Oligodeoxynucleotides containing modified pyrimidine bases which can stably hydrogen-bond to adenine and guanine and also purine bases which pair with thymine and cytosine residues in duplexes have been synthesised, as have oligomers with both such analogues. Structures have been investigated by melting transitions, n.m.r. spectroscopy and crystallography and then interpreted in terms of tautomeric equilibria. Applications to hybridisation probes and primers will be discussed.

Synthesis of oligodeoxyribonucleotides containing degenerate bases and their use as primers in the polymerase chain reaction.

Heptadecaoligodeoxyribonucleotides containing one or more of the bases, 6H,8H-3,4-dihydropyrimido[4,5-c][1,2]oxazin-7-one (P), 2-amino-6-methoxyaminopurine (K), and hypoxanthine (I) and combinations of P with K and I have been synthesised on a DNA synthesiser. The stability of duplexes containing these basemodified oligomers with P/A, P/G, K/C and K/T; P/A, P/G, I/C, I/T and I/A, I/G, I/C, I/T base pairs were compared by measuring their melting transition (Tm) values. Oligomers containing both P and K and P and I were more stable than those with I alone or with mismatches. These oligomers together with one with a P base at the 3'-end were used as primers in polymerase chain reaction (PCR) experiments. They were all effective primers except one with I alone and a triple mismatch. Thus the use of the degenerate bases P and K in primer design is established.

The synthesis and in vitro cytotoxic studies of novel bis-naphthalimidopropyl polyamine derivatives.

Bis-naphthalimidopropyl putrescine (BNIPPut), spermidine (BNIPSpd), spermine (BNIPSpm) and oxa-putrescine (BNIPOPut) were synthesised and their growth-inhibitory properties characterised. All these compounds except for BNIPOPut, showed high in vitro cytotoxic activity (with mean GI50 values between 0.5 and 8.45 microM) and selectivity against cancer cells derived from nine different human tumours. The increased content of nitrogen atoms in the linker chain of BNIPSpd and BNIPSpm significantly improved their aqueous dissolution properties with a marginal decrease in their cytotoxic activity.

Repeated pregnancy without lactation: effects on carcass composition and adipose tissue cellularity in rats.

Four groups of Sprague-Dawley rats, 200-225 g, were mated. Two groups of rats were killed after 3 wk of nursing eight pups (PL-1) or no lactation (PNL-1). The remaining two groups went through three cycles of pregnancy/lactation (PL-3) or pregnancy/no lactation (PNL-3) and were then killed for carcass composition analysis. Two virgin groups (CON-1, CON-3) served as age controls and were killed at the appropriate time. There was a gradual reduction in food intake from cycle 1 to cycle 3 for all groups. PL-3 rats ingested significantly more food than PNL-3 rats in pregnancies of cycle 2 and cycle 3. At the end of cycle 1, there was no difference in body weight, carcass weight, fat content and fat cell cellularity in parametrial and subcutaneous fat pads between three groups of rats. However, after three cycles, the PL-3 group had significantly reduced carcass fat content because of a significant reduction in fat cell number. Repeated pregnancy followed by no lactation resulted in greater carcass fat content and fat cell number in the subcutaneous pad than observed in lactating rats, although these increases failed to reach significance when compared with virgin controls. Thus lactation may not only be beneficial to the offspring but also enhances maternal weight loss and prevents obesity in multiparous individuals.

The synthesis and in vitro cytotoxic studies of novel oxa-spermidine derivatives and homologues.

A series of oxa-spermidine derivatives and homologues were prepared and their anticancer properties were evaluated. All these compounds showed an average GI50 value in the range of 3.9-28.9 microM. SAR studies showed that the presence of a sulphonamido functionality and the length of the alkyl chain are important factors for an enhanced activity.

An intracranial aneurysm associated with a recurrent meningioma--a case report.

A patient presented with late onset seizure due to sphenoid ridge meningioma excision, during a craniotomy. Nine years after craniotomy, sphenoid ridge meningioma recurred in this patient along with middle cerebral artery (MCA) aneurysm. Peripheral intracranial aneurysm has been shown to be caused by many etiologies. But a direct relationship to an intracranial neoplasm is extremely rare. A causal relationship between the growth of the tumor and the development of the aneurysm is postulated.