The effects of prostaglandins E2 and F2α on synaptosomal accumulation and release of 3H-norepinephrine

Prostaglandins (PG) of both the E and F series may serve as modulators of norepinephrine (NE) release from peripheral sympathetic neurons. We have studied the effects of PGE₂ and PGF_2α on the accumulation and release of ³H-NE in the CNS using synaptosomes isolated from rat hypothalami.The release of ³H-NE from synaptosomes superfused with Krebs-Ringer bicarbonate buffer was multiphasic with an initial fast release phase followed by a slower release. Raising KC1 concentration of the superfusion medium to 56mM during the slow release phase is known to stimulate ³H-NE release. PGE₂ (1 × 10^?6M) attenuated ³H-NE release during the fast phase and reduced the amount of ³H-NE released due to KC1 stimulation. At lower concentrations of PGE₂ there was no change in the release profile. PGF_2α was without effect on ³H-NE release at all concentrations tested.The accumulation of ³H-NE was significantly diminished by PGE₂ at a concentration of 1 × 10^?6M, while a lower concentration (1 × 10^?7M) was ineffective. PGF_2α had no effect on ³H-NE accumulation at all concentrations investigated.     

Omega chain methylated analogs of PGF2α and PGE2

Our previously published prostaglandin (PG) synthesis route, in which the ω-chain is added in the penultimate step, provides facile access to a wide variety of ω-chain variant PG analogs. Each series requires only the synthesis of the appropriate methylated acylphosphonate for the Emmons' condensation. The syntheses of analogs bearing the following methylation pattern are detailed: 15-Me; 17, 17-(Me)₂; 17, 17, 20-(Me)₃; 18, 18, 20-(Me)₃; 15, 18, 18, 20-(Me)₄; and 15-Ome-18, 18, 20-(Me)₃. The well-known 16, 16-dimethyl prostaglandins have also been prepared by this sequence. The synthesis of 16, 16-tetramethylene-PG analogs is also described.     

Effect of mifepristone and antiestrogens on uterine PGF2α and PGE2 concentrations in ovariectomized and pregnant rats

Four antiestrogens (anordiol, tamoxifen, RU 39411, ICI 182780) and the antiprogestin, mifepristone (RU 486), were administered to the following three animal models: (1) ovariectomized rats, (2) mated rats treated post-coitally; and (3) pregnant rats treated post-implantation. The antiestrogens were administered alone or in combination with mifepristone at doses effective in preventing and/or terminating pregnancy in rats. The objective of the study was to determine whether these drugs influenced uterine concentrations of prostaglandins (PGF_2α and PGE₂).Antiestrogens administered alone to ovariectomized rats did not effect uterine PGE₂ or PGF_2α concentrations; whereas the combination of anordiol/mifepristone increased uterine PGF_2α concentration, resulting in an increase in the PGF_2α/PGE₂ ratio.Mated rats were treated post-coitally for three consecutive days with anordiol, tamoxifen, estradiol and mifepristone alone and with the combination of anordiol/mifepristone and tamoxifen/mifepristone. An increase in uterine PGF_2α concentrations and in the PGF_2α/PGE₂ ratio occurred only in anordiol/mifepristone treated group. A decrease in uterine PGE₂ concentrations occurred in animals treated with anordiol, tamoxifen and estradiol, resulting in an increase in the PGF_2α/PGE₂ ratio.Anordiol (5.0 mg/kg/day) and mifepristone (4.0 mg/kg/day) alone and the combination of anordiol/mifepristone (2.5/1.0 mg/kg/day) administered to pregnant rats on days 7, 8 and 9 of pregnancy induced an increase in PGF_2α levels without affecting uterine PGE₂ concentration. The changes in uterine PGF_2α concentrations induced by anordiol and the combination of anordiol/mifepristone resulted in an increase in the PGF_2α/PGE₂ ratio.The antiestrogens tested except for ICI 182780 possessed agonist activity when assayed by measuring their capacity to increase the uterine weights in ovariectomized rats. Also, ICI 182789 was the only antiestrogen that did not influence uterine PG concentrations. It can be concluded that ICI 182780 is the only “pure” antiestrogen among those tested.The present results show that antiestrogens and the combination of mifepristone plus anordiol at doses preventing implantation and terminating pregnancy increase uterine PGF_2α and/or decrease PGE₂ concentrations, resulting in an alteration of PGF_2α/PGE₂ ratio. These findings suggest that there exists a critical balance of PGF_2α to PGE₂ concentrations in the uterus required for the normal passage of fertilized ova through the oviduct, initiating implantation of the blastocysts, development of embryos, and maintenance of pregnancy.     

Synthesis and reactions of 1′,2:4,6-di-o-isopropylidene-sucrose

The reaction of sucrose with a combination of 2,2-dimethoxypropane, N,N-dimethylformamide, and toluene-p-sulphonic acid (reagent A) gave, after acetylation followed by chromatography, 1′,2:4,6-di-O-isopropylidenesucrose tetra-acetate (1) in 15% yield. The structure of 1 was determined on the basis of p.m.r. and mass spectrometry, and by chemical transformations. Treatment of 1 with aqueous acetic acid afforded sucrose 3,3′,4′,6′-tetra-acetate 2. Reacetalation of 2 using reagent A gave 1 in 80% yield. The p.m.r. spectrum of 2 confirmed the presence of hydroxyl groups at C-2 and C-4. The following sequence of reactions showed that the remaining two hydroxyl groups were located at C-6 and C-1′. Selective tritylation of 2 gave 1′,6-di-O-tritylsucrose 3,3′,4′,6′-tetra-acetate (3) as the minor, and 6-O-tritylsucrose 3,3′,4′,6′-tetra-acetate (4) as the major, product. When tritylation was carried out under forcing conditions, 2 gave 3 as the major product. Acetylation of 4 afforded 6-O-tritylsucrose hepta-acetate. Mesylation of 2 gave the tetramethanesulphonate 5, which afforded the 6-dcoxy-6-iodo derivative 6 on treatment with a refluxing solution of sodium iodide in butanone. Treatment of 3 with methanesulphonyl chloride in pyridine gave the disulphonate 7, which on detritylation followed by acetylation gave 2,4-di-O-methanesulphonylsucrose hexa-acetate (9). Treatment of 9 with sodium benzoate in hexamethylphosphoric triamide displaced the 4-sulphonate, with inversion of configuration, to give the galacto derivative 10.     

Inheritance of Lysosomal Acid β-Galactosidase Activity and Gangliosides in Crosses of DBA/2J and Knockout Mice

GM1 gangliosidosis is a progressive neurodegenerative disease caused by deficiencies in lysosomal acid beta-galactosidase (beta-gal) and involves accumulation and storage of ganglioside GM1 and its asialo form (GA1) in brain and visceral tissues. Similar to the infantile/juvenile human disease forms, B6/129Sv beta-gal knockout (ko) mice express residual tissue beta-gal activity and significant elevations of brain GM1, GA1, and total gangliosides. Previous studies suggested that inbred DBA/2J (D2) mice may model a mild form of the human disease since total brain ganglioside and GM1 concentration is higher while beta-gal specific activity is lower (by 70-80%) in D2 mice than in inbred C57BL/6J (B6) mice and other mouse strains. A developmental genetic analysis was conducted to determine if the genes encoding beta-gal (Bgl) in the D2 and the ko mice were functionally allelic and if the reduced brain beta-gal activity in D2 mice could account for elevations in total brain gangliosides and GM1. Crosses were made between D2 mice homozygous for the Bgld allele (d/d), and either B6/129Sv mice heterozygous for the Bgl+ allele (+/-) or homozygous for the ko Bgl- allele (-/-) to generate d/+ and d/- mice. Specific beta-gal activity (nmol/mg protein/h) showed additive inheritance in brain, liver, and kidney at juvenile (21 days) and adult (255 days) ages with the d/- mice having only about 16% of the beta-gal activity as that in the +/+ mice. These results indicate that the Bgl genes in the D2 and the ko mice are noncomplementing functional alleles. However, the d/- mice did not express GA1 and had total brain ganglioside and GM1 concentrations similar to those in the d/+ and +/+ mice. These results suggest that the reduced brain beta-gal activity alone cannot account for the elevation of total brain gangliosides and GM1 in the D2 mice.     

Synthesis and Antitumor Activity of Acyl and Benzyl Types of Prodrugs of 2′-Deoxy-2′-methylidenecytidine

Abstract

For the purpose of improvement of the in vivo antitumor activity of 2′-deoxy-2′-methylidenecytidine (DMDC, 1), we synthesized its various acyl and benzyl derivatives and evaluated them for their antitumor activity against P388 murine leukemia in mice. In terms of minimum effective dose (30% increase in life span), 5′-O-stearoyl DMDC showed two-fold higher antitumor activity than DMDC on a molar basis, when intraperitoneally (i.p.) administered to mice once a day. The antitumor activities of some other acyl derivatives were almost comparable to that of DMDC, while benzyl derivatives had no antitumor activity. Results on the hydrolysis of 5′-O-acyl derivatives by porcine liver esterase showed that at least these derivatives should not be resistant to enzymatic hydrolysis for exhibiting antitumor activity. After either an i.p. or oral dose of 3′-O-benzyl DMDC, very low concentrations of blood DMDC were seen compared with those after administration of DMDC, suggesting that the inactivity of benzyl derivatives as prodrugs was due to the minimal level of DMDC in circulation after administration.     

Preparation and Characterization of Oligonucleotides of D- and L-2’ Deoxyuridine

Abstract

The synthesis of oligonucleotides of 2'deoxyuridine containing both the natural D-2'deoxyribose and the unnatural L-2'deoxyribose is described. Units up to the 18-mer have been made via a modified triester procedure and characterized by HPLC.     

Concise and Efficient Synthesis of 3′-O-Tetraphosphates of 2′-Deoxyadenosine and 2′-Deoxycytidine

We describe concise and efficient synthesis of biologically very important 3′-O-tetraphosphates namely 2′-deoxyadenosine-3′-O-tetraphosphate (2′-d-3′-A4P) and 2′-deoxycytidine-3′-O-tetra-phosphate (2′-d-3′-C4P). N⁶-benzoyl-5′-O-levulinoyl-2′-deoxyadenosine was converted into N⁶-benzoyl-5′-O-levulinoyl-2′-deoxyadenosine-3′-O-tetraphosphate in 87% yield using a one-pot synthetic methodology. One-step concurrent deprotection of N⁶-benzoyl and 5′-O-levulinoyl groups using concentrated aqueous ammonia resulted 2′-d-3′-A4P in 74% yield. The same synthetic strategy was successfully employed to convert N⁴-benzoyl-5′-O-levulinoyl-2′-deoxycytidine into 2′-d-3′-C4P in 68% yield.     

Syntheses of 2-acetamido-2-deoxy-4-O-βD-galacto-pyranosyl-D-mannose and -D-glucose and of 2-acetamido-2-deoxy-4-O-βD-mannopyranosyl-D-mannose and -D-glucose

2-acetamido-2-deoxy-4-O-β-D-galactopyranosyl-D-mannose (6) and -D-glucose (7) were prepared by addition of nitromethane to 3-O-β-D-galactopyranosyl-D-arabinose, followed by acetylation, ammonolysis, and application of the Nef reaction. Similarly, 2-acetamido-2-deoxy-4-O-β-D-mannopyranosyl-D-mannose (14) and -D-glucose (15) were prepared by the same scheme from 3-O-β-D-mannopyranosyl-D-arabinose. In the two series of experiments, 6 and 14 were the respective major products. Epimerization of the 2-acetamido-2-deoxy-D-mannose residue in 6 and 14 yielded 7 and 15, respectively.     

Origin and early evolution of eukaryotes inferred from the amino acid sequences of translation elongation factors 1α/Tu and 2/G

Phylogenetic placements of archaebacteria and protozoa are important in understanding the origin and early evolution of eukaryotes. These problems have been analyzed mainly by comparisons of small subunit ribosomal RNA (SrRNA) sequences. However, the SrRNA phylogeny may sometimes be unreliable, especially when base compositions are biased among species. Because it is difficult to take full account of the bias in inferring the SrRNA tree, alternative examinations using protein sequence data have been very much desired. We analyzed the phylogenetic relationship among eukaryotes, archaebacteria, and eubacteria by the ML method of protein phylogeny using amino acid sequence data of EF-1α/Tu and 2/G. The unrooted tree analyses of both the EF-1α/Tu and 2/G consistently demonstrated that the ‘eocyte’ tree, in which archaebacteria are not monophyletic but eocytes are closer to eukaryotes than to other archaebacteria, is very likely. Further analysis using a composite tree of EF-1α/Tu and 2/G suggested that archaebacteria are closer to eukaryotes than to eubacteria but are not monophyletic. These results clearly support the hypothesis that eukaryotes have evolved from the eocyte-like organism. We also analyzed a protozoan phylogeny including mitochondrion-lacking species by the ML method using EF-1α and EF-2 data sets, and demonstrated (a) that two mitochondrion-lacking species, G. plecoglossi (Microsporidians) and G. lamblia (Diplomonads) probably represent the first and the second earliest offshoots of eukaryotes, respectively; (b) that Trypanosoma is not likely to have diverged next to Giardia as suggested by the SrRNA tree, but shows high affinity with higher eukaryotes; and (c) that protein phylogeny would give a robust estimation because amino acid compositions of conservative proteins do not differ significantly among species.     

Addition of deoxycholate in electroimmunoassay and crossed immunofocusing for quantification of β2-glycoprotein I and its subfractions

β₂-GlycoproteinI was shown to be a hydrophilic protein exhibiting no charge shift in the presence of a cationic detergent, but a charge shift in the presence of an anionic detergent. The latter was suggested to be caused by a binding of β₂-glycoprotein I to deoxycholate in the Triton X-100/deoxycholate micelles. Quantification by electroimmunoassay of asialo-β₂-glycoprotein and I and subfractions of β₂-glycoprotein I gave different results although identical results were obtained in single radial immunodiffusion. Addition of 0.2% (w/v) of deoxycholate to the agarose gels containing Triton X-100 prior to electrophoresis, however, eliminated these differences. The effect of deoxycholate on the rate of migration of β₂-glycoprotein I was found applicable for electroimmunoassay of the protein. Crossed immunofocusing of plasma from individual donors, electrophoresed in an agarose containing Triton X-100/deoxycholate micelles confirmed a postulated variation in the relative composition of subfractions of β₂-glycoprotein I in plasma earlier suggested by Finlayson and Mushinski (Finlayson, J.S. and Mushinski, J.F. (1967) Biochim. Biophys. Acta 147, 413–420).     

Synthesis and evaluation of γ-lactam analogs of PGE₂ as EP4 and EP2/EP4 agonists

To identify topically effective EP4 agonists and EP2/EP4 dual agonists with excellent subtype selectivity, further optimization of the 16-phenyl ω-chain moiety of the γ-lactam 5-thia prostaglandin E analog and the 2-mercaptothiazole-4-carboxylic acid analog were undertaken. Rat in vivo evaluation of these newly identified compounds as their poly (lactide-co-glycolide) microsphere formulation, from which sustained release of the test compound is possible, led us to discover compounds that showed efficacy in a rat bone fracture healing model after its topical administration without serious influence on blood pressure and heart rate. A structure-activity relationship study is also presented.     

Comparison of the effects of prostaglandins D2, F2α and E1 on spontaneous contractions of rabbit oviduct

The effects of PGD₂, PGF_2α and PGE₁ were studied on the circular muscle of post-ovulatory rabbit oviducts $\text{in vitro}$. PGE₁ inhibited spontaneous contractile activity. Lower concentrations of PGD₂ and PGF_2α were stimulatory and higher concentrations were inhibitory. Since PGD₂ may be produced in the oviduct, any hypothesis concerning the role of prostaglandins in the control of oviductal motility and ovum transport should include PGD₂ as well as PGFs and PGEs.     

Effects of α-adrenoceptor antagonists on ABCG2/BCRP-mediated resistance and transport

Acquired resistance of cancer cells to various chemotherapeutic agents is known as multidrug resistance, and remains a critical factor in the success of cancer treatment. It is necessary to develop the inhibitors for multidrug resistance. The aim of this study was to examine the effects of eight α-adrenoceptor antagonists on ABCG2/BCRP-mediated resistance and transport. Previously established HeLa/SN100 cells, which overexpress ABCG2/BCRP but not ABCB1/MDR1, were used. The effects of the antagonists on sensitivity to mitoxantrone and the transport activity of Hoehst33342, both substrates for ABCG2/BCRP, were evaluated using the WST-1 assay and cellular kinetics, respectively. ABCG2/BCRP mRNA expression and the cell cycle were also examined by real-time RT-PCR and flow cytometry, respectively. Sensitivity to mitoxantrone was reversed by the α-adrenoceptor antagonists in a concentration-dependent manner, although such effects were also found in the parental HeLa cells. Levels of ABCG2/BCRP mRNA expression were not influenced by the antagonists. The transport activity of Hoechst33342 was decreased by doxazosin and prazosin, but unaffected by the other antagonists. In addition, doxazosin and prazosin increased the proportion of S phase cells in the cultures treated with mitoxantrone, whereas the other α-adrenoceptor antagonists increased the percentage of cells in G(2)/M phase. These findings suggested that doxazosin and prazosin reversed resistance mainly by inhibiting ABCG2/BCRP-mediated transport, but the others affected sensitivity to mitoxantrone via a different mechanism.     

In vitro incorporation of 2′-deoxyadenosine and 3′-deoxyadenosine into yeast tRNAPhe using tRNA nucleotidyl transferase,and properties of tRNAPhe-C-C-2′dA and tRNAPhe-C-C-3′dA

2′-Deoxyadenosine and 3′-deoxyadenosine (cordycepin) can be incorporated into the 3′-terminal position of tRNA^Phe by tRNA nucleotidyl transferase. tRNA^Phe-C-C-2′dA and tRNA^Phe-C-C-3′dA, missing the cis-diol group at the 3′-terminal end are resistant to periodate oxidation and are not able to form borate complexes. In aminoacylation experiments only the tRNA^Phe-C-C-3′dA proved to be chargeable.     

2-Pyridylmetallocenes: Part I. Electrophilic halogenation of 2-pyridylferrocene. Molecular structures of 2-pyridylferrocene and its α-brominated and -fluorinated derivatives. Synthesis of 2-pyridylruthenocene and 2-pyridylcymantrene

A new high-yield synthesis of 2-pyridylferrocene (1) without formation of the 1,1′-disubstituted product has been developed. Also the corresponding ruthenocene and cymantrene derivatives [C₅H₄(2-C₅H₄N)]ML_n (ML_n = Ru(C₅H₅) (2), Mn(CO)₃ (3)) were prepared and fully characterized. Ortho-lithiation of 1 followed by electrophilic halogenation yielded [C₅H₃X(2-C₅H₄N)]Fe(C₅H₅) [X = F (4), Cl (5), Br (6), I (7)], with 4 only being the second reported and first fully characterized fluoroferrocene. The molecular structures of 1, 4 and 6 have been determined by X-ray crystallography.     

Physical and functional interaction of the active zone protein CAST/ERC2 and the β-subunit of the voltage-dependent Ca(2+) channel

In the nerve terminals, the active zone protein CAST/ERC2 forms a protein complex with the other active zone proteins ELKS, Bassoon, Piccolo, RIM1 and Munc13-1, and is thought to play an organizational and functional role in neurotransmitter release. However, it remains obscure how CAST/ERC2 regulates the Ca(2+)-dependent release of neurotransmitters. Here, we show an interaction of CAST with voltage-dependent Ca(2+) channels (VDCCs), which are essential for regulating neurotransmitter release triggered by depolarization-induced Ca(2+) influx at the active zone. Using a biochemical assay, we showed that CAST was coimmunoprecipitated with the VDCC β(4)-subunit from the mouse brain. A pull-down assay revealed that the VDCC β(4)-subunit interacted directly with at least the N- and C-terminal regions of CAST. The II-III linker of VDCC α(1)-subunit also interacted with C-terminal regions of CAST; however, the interaction was much weaker than that of β(4)-subunit. Furthermore, coexpression of CAST and VDCCs in baby hamster kidney cells caused a shift in the voltage dependence of activation towards the hyperpolarizing direction. Taken together, these results suggest that CAST forms a protein complex with VDCCs, which may regulate neurotransmitter release partly through modifying the opening of VDCCs at the presynaptic active zones.     

The roles of PGF2α and PGE2 in regression of the corpus luteum after intrauterine infusion of Arcanobacterium pyogenes in cows

To study the effect of bacteria in the uterus on the fate of the corpus luteum (CL), Arcanobacterium pyogenes was inoculated into the uteri of cows on Day 3 (Day 0 = day of spontaneous ovulation). Plasma concentrations of 13,14-dihydro-15-keto-PGF_2α (PGFM), 13,14-dihydro-15-keto-PGE₂ (PGEM) and progesterone (P₄) were determined. In five cows, the developing CL regressed and first-wave dominant follicles, which normally become atretic, ovulated (Group OV) after bacterial inoculation. In another five cows (Group NOV) and five control cows, the developing CL did not regress and first-wave dominant follicles did not ovulate. In Group OV, PGFM concentrations increased by 126.2 pg/mL (from 36.8 ± 7.8 pg/mL on Day 3 to 163 ± 37.2 pg/mL on Day 6), with an increase ratio of 5.8-fold. Conversely, in Group NOV, PGFM had a greater increase of 198.4 pg/mL (from 128.2 ± 27.8 pg/mL on Day 3 to 326.6 ± 115.1 pg/mL on Day 5), but the increase ratio was only 2.3-fold. Although PGEM tended to increase in both groups, raw increases and increase ratios were small. Bacterial inoculation into the uterus stimulated the release of prostaglandins and affected the fate of the CL; in that regard, the CL was affected more by PGF_2α than by PGE₂, and the increase ratio of PGF_2α was more important than the raw increase.