Palmitic and stearic acids similarly affect plasma lipoprotein metabolism in cynomolgus monkeys fed diets with adequate levels of linoleic acid.

This study was designed to evaluate whether the exchange of specific saturated fatty acids [SFA; palmitic acid (16:0) for stearic acid (18:0)] would differentially affect plasma lipids and lipoproteins, when diets contained the currently recommended levels of total SFA, monounsaturated fatty acids and polyunsaturated fatty acids (PUFA). Ten male cynomolgus monkeys were fed one of two purified diets (using a cross-over design) enriched either in 16:0 (palmitic acid diet) or 18:0 (stearic acid diet). Both diets provided 30% of energy as fat (SFA/monounsaturated fatty acid/PUFA: 1/1/1). The palmitic acid and stearic acid diets were based on palm oil or cocoa butter (59% and 50% of the total fat, respectively). By adding different amounts of sunflower, safflower and olive oils, an effective exchange of 16:0 for 18:0 of approximately 5% of energy was achieved with all other fatty acids being held constant. Monkeys were rotated through two 10-wk feeding periods, during which time plasma lipids and in vivo lipoprotein metabolism (following the simultaneous injection of (131)I-LDL and (125)I- HDL were evaluated). Plasma triacyglycerol (0.40 +/- 0.03 vs. 0.37 +/- 0.03 mmol/L), plasma total cholesterol (3.59 +/- 0.18 vs. 3.39 +/- 0.23 mmol/L), HDL cholesterol (1.60 +/- 0.16 vs 1.53 +/- 0.16 mmol/L) and non-HDL cholesterol (2.02 +/- 0.26 vs. 1.86 +/- 0.23 mmol/L) concentrations did not differ when monkeys consumed the palmitic acid and stearic acid diets, respectively. Plasma lipoprotein compositional analyses revealed a higher cholesteryl ester content in the VLDL fraction isolated after consumption of the stearic acid diet (P < 0.10), as well as a larger VLDL particle diameter (16.3 +/- 1.7 nm vs. 13.8 +/- 3.6 nm; P < 0.05). Kinetic analyses revealed no significant differences in LDL or HDL transport parameters. These data suggest that when incorporated into diets following current guidelines, containing adequate PUFA, an exchange of 16:0 for 18:0, representing approximately 11 g/(d.10.46 mJ) [ approximately 11 g/(d.2500 kcal)] does not affect the plasma lipid profile and has minor effects on lipoprotein composition. Whether a similar effect would occur in humans under comparable dietary conditions remains to be established.

Pork fat and chicken fat similarly affect plasma lipoprotein metabolism in cynomolgus monkeys fed diets with adequate levels of linoleic acid.

The effects on plasma lipoprotein metabolism of replacing pork fat (PF) with chicken fat (CF) (formulated as part of currently recommended prudent diets) was evaluated in 10 male cynomolgus monkeys. Monkeys were rotated through three dietary periods, (each of 10-wk duration), during which total cholesterol (TC), triacylglycerol (TG) and HDL-cholesterol (HDL-C) were measured (7, 8 and 9 wk) and in vivo lipoprotein metabolism evaluated (after 9 wk). Initially, all monkeys were fed a high-fat, high-cholesterol reference diet [38% of energy (en) from fat, 18%en saturated fatty acids (SFA), 10%en monounsaturated fatty acids (MUFA), 10%en polyunsaturated fatty acids (PUFA), 0.045 mg cholesterol/kJ diet]. Subsequently, monkeys were rotated through two test diets (30%en fat, SFA/MUFA/PUFA 1:1:1, 0.004-0.005 mg cholesterol/kJ diet), in which 80% of the fat was either PF or CF, with the remaining 20% derived from high-linoleic safflower oil. There was no significant difference between the two test diets for TG, TC, nonHDL-C, HDL-C or the ratio of TC/HDL-C. Lipoprotein composition, LDL apolipoprotein B pool size, fractional catabolic rate and transport rate were also not significantly different when monkeys consumed the two test diets. These data suggest that when incorporated into diets following current guidelines and containing adequate PUFA ( approximately 7-9%en), PF and CF similarly affect plasma lipids.

Effects of exchanging 4%en between dietary stearic and palmitic acid on hamster plasma lipoprotein metabolism.

This study was designed to determine whether the exchange of specific fatty acids (palmitic (16:0) for stearic (18:0)), would exert differential effects on plasma and lipoprotein lipids, when diets contained approximately 30%en from fat with adequate levels of linoleic acid (18:2). Thirty-two male Golden Syrian hamsters were fed isocaloric purified diets with comparable amounts of 18:2 (approximately 10.5%en). The 18:0-rich diet (50% cocoa butter, 41% safflower oil, 9% sunflower oil) provided 4.8%en 16:0 and 5.3%en from 18:0, while the 16:0-rich diet (59% palm oil, 36% safflower oil, 5% olive oil) provided 8.7%en from 16:0 and 1.2%en from 18:0, resulting in a 16:0/18:0 exchange of approximately 4%en. Both diets contained negligible amounts of lauric and myristic acid (< 0.2%en), approximately 9.5%en from oleic acid and 77 mg cholesterol/1000 kcal. Animals were fed their respective diets for 4 weeks at which point various lipid and lipoprotein parameters were measured. There were no significant difference between dietary groups for any of the measured parameters, which included body weights, food consumption, plasma lipids, lipoprotein lipid and apoprotein concentrations, as well as lipoprotein compositions. Additionally, estimated diameters of various lipoprotein particles were not affected by the fatty acid exchanges employed. Thus these data suggest that when total fat is restricted to 30%en and 18:2 levels are approximately 10%en, a 4%en exchange between 16:0 and 18:0 (representing intakes of approximately 9 g/d/2000 kcal diet) produces comparable plasma lipids.

N,N-dimethyl-5-methoxymethyl-2'-deoxycytidine.

In the title compound, C13H21N3O5, the pyrimidine ring adopts the antiperiplanar (-ap) conformation [chi = 193.54 (19) degrees]. The deoxyribose sugar ring has the C2'-exo-C3'-endo (2T3) twist conformation. The pseudo-rotational parameters of the deoxyribose sugar ring are P = 6.83 (2) degrees and Tm = 38.27 (2) degrees. The exocyclic side chain at C5' has the g+ conformation [gamma = 47.7 (3) degrees]. The 5-methoxymethyl group is distal to the deoxyribose sugar ring, with a C6-C5-C52-O52 torsion angle of -91.9 (3) degrees.

2',3'-Didehydro-2',3'-dideoxy-5-hydroxymethyluridine.

The furanose ring in C10H12N2O5 adopts the O(4')-endo envelope conformation (0E) and the glycosidic torsion angle C(2)--N(1)--C(1')--O(4'), chi, is 245.2 (3) degrees. The pseudo-rotational parameters are P = 102.7 degrees and tau m = 5.2 degrees. The CH2OH group on C(5') has the t conformation [gamma = 179.2 (2) degrees].

Modified tetrazolium-based colorimetric method for determining the activities of anti-HIV compounds.

A modified tetrazolium-based colorimetric assay was used to determine the anti-HIV activities of ddAzThd, ddCyd, ddIno, and PFA. In this assay, poly-1-lysine-coated plates were used to attach the MT-2 cells to the bottom of the plates. A fixed amount of virus (50 TCID50) was used in each well. A modified version of the formula published by Pauwels et al. (1988) was used for calculating the percentage cell protection from virus infection. Using CC10/EC90 to calculate the selective indices, the decreasing order of selectivity against HIV-1 strain A87SF, was: ddAzThd greater than PFA greater than ddCyd greater than ddIno. Against HIV-1 strain A79SK-1 the decreasing order of selectivity was: PFA greater than ddIno greater than AzThd greater than ddCyd. The modified formula showed lack of anti-HIV activity for thymidine at non-toxic concentrations.

Deoxyribonucleoside triphosphate pools of herpes simplex virus infected cells: the influence of selective antiherpes agents and the role of the deaminase pathway.

The effect of 5-methoxymethyl-2'-deoxycytidine (MMdCyd), in combination with tetrahydrodeoxyuridine (H4dUrd) and 5-methoxymethyl-2'-deoxyuridine (MMdUrd) on deoxyribonucleoside triphosphate pools was assessed. The dNTP pool content was almost 5 times as high in herpes simplex virus (HSV) infected VERO cells compared with mock-infected cells. Significant differences in dNTP pool sizes were observed with the different treatments. Treatment of HSV-infected cells with MMdCyd and MMdUrd resulted in a massive expansion of the dTTP pool, whereas pools of dCTP and dGTP were not affected substantially. MMdUrd and MMdCyd produced dATP pools that were 4 and 2.5 times that of the controls, respectively. Treatment with H4dUrd resulted in the dCTP pool increasing 12 times and barely detectable levels of dTTP. MMdCyd in combination with H4dUrd resulted in a marked reduction of the total deoxyribonucleoside triphosphate level. These results indicate that during viral replication the bulk of the thymidine nucleotides are derived from the dCyd/dCMP deaminase de novo pathway.

Regulatory effects of deoxyribonucleosides on the activity of 5-methoxymethyl-2'-deoxycytidine: modulation of antiherpes activity by deoxyguanosine and tetrahydrodeoxyuridine.

The effect of purine and pyrimidine deoxyribonucleosides on the activity of 5-methoxymethyl-2'-deoxycytidine (MMdCyd) against herpes simplex virus type 1 (HSV-1) was investigated. The antiviral activity of MMdCyd was decreased by deoxythymidine, deoxyuridine and deoxycytidine. Deoxyadenosine had no effect at concentrations up to 500 microM. In contrast, deoxyguanosine (dGuo) potentiated MMdCyd activity. The mean ED50 (1.5 microM) for the combination (MMdCyd plus 100 microM dGuo) was approximately 20-fold lower than that of MMdCyd (ED50 26 microM). When tetrahydrodeoxyuridine (H4dUrd, 540 microM) was added along with MMdCyd and dGuo, anti-HSV-1 activity of MMdCyd was further potentiated by 25-fold (ED50 0.06 microM). The inhibition of virus replication, as determined by the plaque reduction assay, was further confirmed by virus yield studies and by parallel observations on virus-induced cytopathogenicity. The order of decreasing effectiveness for reducing the production of infectious virus particles (virus yield) by different treatments was: MMdCyd + dGuo + H4dUrd greater than MMdCyd + DGuo greater than MMdCyd + H4dUrd greater than MMdCyd greater than dGuo + H4dUrd greater than dGuo greater than H4dUrd. The effect of dGuo and dGuo in combination with H4dUrd on deoxyribonucleoside triphosphate (dNTP) pools was determined in Vero cells infected with multiplicity of infection of 5 PFU/cell. In the presence of 100 microM dGuo, there was approximately a 3-fold, 2-fold and 12-fold increase in dCTP, dTTP and dGTP pool sizes respectively, as compared to control (untreated) cells. Treatment with H4dUrd (1.06 mM) in combination with dGuo (100 microM), resulted in an increase of the dCTP pool and a marked fall in the dTTP and dGTP pool. The possible mechanisms for potentiation of MMdCyd activity by dGuo and H4dUrd are discussed.

Structure and conformation of 5-methoxymethyl-N4-methyl-2'-deoxycytidine.

C12H19N3O5, Mr = 285.25, monoclinic, P2(1), a = 7.0180 (6), b = 8.6946 (11), c = 10.7715 (10) A, beta = 91.055 (7) degrees, V = 657.15 A3, Z = 2, Dx = 1.441 g cm-3, lambda(Cu K alpha) = 1.5418 A, mu = 9.63 cm-1, F(000) = 304, T = 287 K, R = 0.039 for 1424 observed reflections. The furanose ring adopts the C(1')-exo envelope conformation (E1), with the glycosyl linkage anti (chi = 193.8 degrees). The pseudo-rotational parameters are P = 130.9 degrees and tau m = 39.4 degrees. In the deoxyribose ring, the side chain on C(5') has the t conformation. In the pyrimidine ring the N4-methyl takes a cis conformation to N(3) and the methoxymethyl side chain is on the same side of the cytidine plane as O(4').

Antiherpes virus activity and effect on deoxyribonucleoside triphosphate pools of (E)-5-(2-bromovinyl)-2'-deoxycytidine in combination with deaminase inhibitors.

The antiviral activity and cytotoxicity of (E)-5-(2-bromovinyl)-2'-deoxycytidine (BrVdCyd) against herpes simplex virus type 1 (HSV-1), singly and in combination with deaminase inhibitors was determined using rabbit kidney (RK-13), HEP-2, BHK-21 and VERO cells. BrVdCyd was a potent inhibitor of HSV-1 replication with ED50 values of 0.30 to 1.20 microM depending on the cell line used. In the presence of tetrahydrouridine or tetrahydrodeoxyuridine (H4dUrd), potency of BrVdCyd increased approximately two fold (ED50: 0.54 microM) in HSV-infected VERO cells. The combination of BrVdCyd and H4dUrd was also effective in decreasing virus yield. Dihydrodeoxyuridine (H2dUrd) reversed the activity of BrVdCyd (ED50: 6 to 7 microM). The effect of (E)-5-(2-bromovinyl)-2'-deoxyuridine (BrVdUrd), BrVdCyd and BrVdCyd in combination with H4dUrd on deoxyribonucleoside triphosphate (dNTP) pools was assessed in VERO cells infected with a high multiplicity of infection (10 PFU/cell). Significant differences in dNTP poll sizes (pmol/10(6) cell) were observed with different treatments. BrVdUrd and BrVdCyd treatment resulted in marked expansion of the dTTP pool (greater than 1200 pmol) compared to HSV-infected VERO cells (303 pmol). Exposure to H4dUrd resulted in a 12-fold expansion of the dCTP pool (326 pmol) and barely detectable levels of dTTP (less than 1.0 pmol). BrVdCyd plus H4dUrd treatment resulted in a slight expansion of the dTTP pool (515 pmol). These results indicate: (i) H4dUrd inhibits de novo dCyd/dCMP deaminase pathway and (ii) exposure to BrVdCyd plus H4dUrd puts a strain on viral DNA synthesis to such an extent that even though dTTP is being formed from alternative pathways, its eventual utilization as a substrate is reduced and hence it builds up.

Structure and conformation of 3'-O-acetyl-2'-deoxy-5-methoxymethyluridine.

C13H18N2O7, Mr = 314.297, triclinic, P1, a = 6.0321 (4), b = 6.775 (5), c = 9.6699 (7) A, alpha = 76.917 (6), beta = 78.871 (6), gamma = 75.344 (6) degrees, V = 368.54 A3, Z = 1, Dm = 1.43, Dx = 1.416 g cm-3, Cu K alpha radiation (Ni filtered), lambda = 1.5418 A, F(000) = 166, T = 287 K, final conventional R factor = 0.034, wR = 0.044 for 1359 reflections and 268 variables. The structure was solved using the XTAL system. The conformation of the furanose ring is best described as intermediate between 2E and 2(1)T: the pseudorotational parameters are P = 148.9 degrees and tau m = 33.4 degrees. The CH2OH, C(5'), side chain has the g+ conformation, the carbonyl bond of the 3'-acetoxy group is syn to the C(3')-O(3',1) bond on the sugar ring and the glycosidic bond conformation is anti [chi = -137.6 (3) degrees]. The methoxy group of the 5-methoxymethyl substituent is on the same side of the pyrimidine plane as O(4') of the furanose ring. Comparison with 2'-deoxy-5-methoxymethyluridine shows that intermolecular attraction have little effect on the internal conformations of the molecule in the solid state.

The alternating conformation of analogues of poly[d(AT)].

Synthetic DNAs were prepared containing 6-methyl adenine (m6A) in place of adenine and 5-ethyl uracil (Et5U) or 5-methoxymethyl uracil (Mm5U) in place of thymine. All three modifications destabilized duplex DNAs to varying degrees. The binding of ethidium was studied to analogues of poly[d(AT)]. There was no evidence of cooperative binding and the "neighbour exclusion rule" was obeyed in all cases although the binding constant to poly[d(m6AT)] was approximately 6 fold higher than to poly[d(AT)]. 31P NMR spectra were recorded in increasing concentrations of CsF. Poly[d(AEt5U)] showed two well-resolved signals separated by 0.55 ppm in 1 M CsF compared to 0.32 ppm for poly[d(AT)] under identical conditions. In contrast, poly[d(AMm5U)] and poly[d(m6AT)] showed two signals separated by 0.28 ppm and 0.15 ppm respectively, only when the concentration of CsF was raised to 2 M. The signals for poly[d(AT)] in 2 M CsF were better resolved and were separated by 0.41 ppm. These results suggest that minor modifications to the bases may have conformational effects which could be recognized by DNA-binding proteins.

Relationship between structure and antiviral activity of 5-methoxymethyl-2'-deoxyuridine and 5-methoxymethyl-1-(2'-deoxy-beta-D-lyxofuranosyl)uracil.

5-Methoxymethyl-1-(2'-deoxy-beta-D-lyxofuranosyl)uracil (MMdLU) was not active against the herpes simplex viruses. The relationship between molecular conformation and antiviral activity for the two epimers, 5-methoxymethyl-2'-deoxyuridine (MMdUrd) and MMdLU, is discussed. MMdUrd was phosphorylated by the virus-induced deoxythymidine kinase. In contrast, MMdLU did not serve as a substrate for the kinase. The geometry and distance between the 5'-CH2OH and 3'-OH groups of the furanose ring appear to be key factors in determining the efficiency of phosphorylation by the virus-induced deoxythymidine kinase, and hence antiviral activity.

Comparison of the mutagenic activity of 5-hydroxymethyldeoxyuridine with 5-substituted 2'-deoxyuridine analogs in the Ames Salmonella/microsome test.

4 antiviral drugs 5-hydroxymethyldeoxyuridine (HMUdR), 5-trifluorothymidine (F3TdR), 5-methoxymethyldeoxyuridine (MMUdR) and 5-ethyldeoxyuridine (EtUdR) have been evaluated for mutagenic activity in the Ames Salmonella/microsome test. The antimetabolites F3TdR and HMUdR were mutagenic in a dose-dependent manner in strain TA100. F3TdR also was mutagenic in strain TA1535. Rat-liver post-mitochondrial supernatant (S9) was not required for mutagenicity.

Efficacy of 5-methoxymethyl-2'-deoxyuridine in combination with arabinosyladenine for the treatment of primary herpes simplex genital infection of mice and guinea pigs.

The relative efficacy of 5-methoxymethyl-2'-deoxyuridine (MMdUrd), arabinosyladenine (ara-A) and the combination of MMdUrd and ara-A in the treatment of experimental genital herpes (GH) was investigated using mouse and guinea pig models. The infection was initiated by intravaginal inoculation using either HSV-2, strain X-265 or HSV-2, strain MS. Treatment was initiated 3 h post virus inoculation. The parameters used to evaluate efficacy were: percent mortality; mean day of death; virus yield from the vaginal secretions; and mean lesion score. The simultaneous application of 5% MMdUrd and 5% ara-A was an effective treatment for controlling primary GH in both animal models. Combination chemotherapy was also effective in preventing recurrence of infection as well as the emergence of drug resistant virus. At 20% concentration, ara-A was effective in providing protection against GH. However, lesions due to recurrent GH appeared after cessation of treatment and the virus isolated from vaginal secretions of ara-A treated animals required higher concentration of drug for inhibition of virus replication in cell culture. 20% MMdUrd was only partially effective in controlling GH. The production of infectious virus particles (virus yield) in cell culture after exposure to either ara-A of MMdUrd alone or in combination was determined. When MMdUrd and ara-A were used together, a substantially lower amount of each drug was needed to inhibit virus production completely and removal of drugs did not result in an increase in virus yield.

Combination chemotherapy: interaction of 5-methoxymethyldeoxyuridine with trifluorothymidine, phosphonoformate and acycloguanosine against herpes simplex viruses.

Methoxymethyldeoxyuridine (MMUdR) when used in combination with either trifluorothymidine (F3TdR) or phosphonoformate (PFA) showed synergistic activity against herpes simplex virus type 1 and type 2 (HSV-1 and HSV-2) in vitro, whereas MMUdR and acycloguanosine (ACG) combination was antagonistic against herpes viruses. HSV-1 mutants resistant to ACG, arabinofuranosyladenine (Ara-A), MMUdR or PFA were isolated. Drug-resistant HSV-1 virus mutants were analyzed for cross sensitivity to ACG, Ara-A, F3TdR, MMUdR, MMUdR-5'-monophosphate (MMUdR-MP) and PFA. The Ara-A-resistant (Ara-AR) virus exhibited 3-fold resistance to MMUdR-MP (ID50 = 105 microM). The ACG-resistant (ACGR) mutant was 160-fold less sensitive to MMUdR (ID50 greater than 1138 microM). The MMUdR-resistant (MMUdRR) mutant remained sensitive to all other antiviral drugs in vitro. Ara-A provided protection against HSV-1 encephalitis in immunosuppressed mice inoculated with a low dose (200 PFU/mouse) of MMUdRR virus or wild-type HSV-1. F3TdR decreased incorporation of tritiated deoxyuridine [( 3H]UdR) in RK-13 cells by 50% at 0.068 microM. Under similar conditions, MMUdR (up to 600 microM) and PFA (up to 208 microM) were without effect on incorporation of [3H]UdR into DNA. In combination chemotherapy experiments, MMUdR (up to 300 microM) used along with F3TdR (up to 1.08 microM) neither decreased nor enhanced cytotoxicity of F3TdR as measured by incorporation of [3H]UdR into cellular DNA. Similarly, MMUdR (up to 300 microM) in combination with PFA (up to 166 microM) was nontoxic to host cells.

Purification and characterization of dihydrofolate reductase from Walker 256 carcinosarcoma.

Dihydrofolate reductase of Walker 256 carcinosarcoma has been purified to homogeneity by affinity chromatography. The enzyme reduced 28 mumol dihydrofolate (FAH2) . min-1 . mg protein-1 at 22 degrees C and pH 7.3. Km values with respect to FAH2 and NADPH were 21 and 29 mM, respectively. The IC50 (amount of inhibitor required for 50% loss of enzyme activity) values were 0.2 nM for MTX and aminopterin and 50 and 67 nM, respectively, for N10-formyl FA and triazinate (NSC-139105). The pH maximum is around pH 7.0 and the isoelectric point is 6.8. This reductase has an apparent molecular weight of 21 500. The N-terminal amino acid is valine and the comparison of the N-terminal 20 residues of this reductase shows very high sequence homology with other mammalian reductases. The enzyme contains two cysteine residues and one of these residues is not involved in catalysis. This reductase has four tryptophan residues and modification of one of these residues leads to loss of activity. The intrinsic circular dichroic (CD) spectrum of this reductase is very different from the CD spectra of reductase of Escherichia coli B and L1210/MTX. However, the CD spectra of the enzyme--substrate and enzyme--inhibitor complexes are very similar to that of the L1210/MTX enzyme. This suggests that the ligands may be constrained in similar conformation on the two enzymes. The fluorescence emission maximum at 314 nM when activated at 286 nM is considerably lower than other mammalian enzymes.