Effects of 1 gram oral or intravenous aspirin on urinary excretion of thromboxane B2 and 6-keto-PGF1α in healthy subjects

Aspirin inhibits cyclo-oxygenase, thus preventing prostanoids formation. After oral administration aspirin is hydrolysed to inactive salicylate partly within the gastrointestinal tract, partly during first pass in the liver, partly in the circulation by plasma esterases. Intravenous aspirin, in contrast, mainly undergoes plasma esterase-catalysed deacetylation. Six healthy male subjects were given 1 g aspirin orally and intravenously two weeks apart according to a cross-over randomized design. Whereas serum T×B2 generation reflecting platelet cyclo-oxygenase activity was suppressed by aspirin by both routes, urinary excretion of T×B2 and 6-keto-PGF1α was not affected by oral aspirin, but was partially though significantly reduced by the i.v. drug. Drug disposition seems therefore to be essential in determining the “biochemical selectivity” of aspirin as related to platelet and renal prostanoids generation.     

Opposing actions of the progesterone metabolites, 5α-dihydroprogesterone (5αP) and 3α-dihydroprogesterone (3αHP) on mitosis,apoptosis, and expression of Bcl-2, Bax and p21 in human breast cell lines

Previous studies have shown that breast tissues and breast cell lines convert progesterone (P) to 5α-dihydroprogesterone (5αP) and 3α-dihydroprogesterone (3αHP) and that 3αHP suppresses, whereas 5αP promotes, cell proliferation and detachment. The objectives of the current studies were to determine if the 5αP- and 3αHP-induced changes in cell numbers are due to altered rates of mitosis and/or apoptosis, and if 3αHP and 5αP act on tumorigenic and non-tumorigenic cells, regardless of estrogen (E) and P receptor status. The studies were conducted on tumorigenic (MCF-7, MDA-MB-231, T47D) and non-tumorigenic (MCF-10A) human breast cell lines, employing several methods to assess the effects of the hormones on cell proliferation, mitosis, apoptosis and expression of Bcl-2, Bax and p21. In all four cell lines, 5αP increased, whereas 3αHP decreased cell numbers, [³H]thymidine uptake and mitotic index. Apoptosis was stimulated by 3αHP and suppressed by 5αP. 5αP resulted in increases in Bcl-2/Bax ratio, indicating decreased apoptosis; 3αHP resulted in decreases in Bcl-2/Bax ratio, indicating increased apoptosis. The effects of either 3αHP or 5αP on cell numbers, [³H]thymidine uptake, mitosis, apoptosis, and Bcl-2/Bax ratio, were abrogated when cells were treated simultaneously with both hormones. The expression of p21 was increased by 3αHP, and was unaffected by 5αP. The results provide the first evidence that 5αP stimulates mitosis and suppresses apoptosis, whereas 3αHP inhibits mitosis and stimulates apoptosis. The opposing effects of 5αP and 3αHP were observed in all four breast cell lines examined and the data suggest that all breast cancers (estrogen-responsive and unresponsive) might be suppressed by blocking 5αP formation and/or increasing 3αHP. The findings further support the hypothesis that progesterone metabolites are key regulatory hormones and that changes in their relative concentrations in the breast microenvironment determine whether breast tissues remain normal or become cancerous.     

Residues of thiamethoxam, aldicarb and its metabolites in coffee leaves and effect on the control of Leucoptera coffeella (Guérin-Mèneville) (Lepidoptera: Lyonetiidae)

The coffee leaf miner Leucoptera coffeella (Guérin-Mèneville), one of the major pests of coffee crops in Brazil, is mainly controlled with insecticides. The objective of this study was to evaluate the residues and the translocation of the insecticide thiamethoxam in coffee leaves, as well as to study its effect on the coffee leaf miner control, comparing it with aldicarb, used as standard. One experiment was set up in the county of Gar?a, SP from December/2001 to August/2002. The treatments used were: aldicarb 150 G at the rates of 2.25 and 4.50 g a.i./pit, thiamethoxam 10 GR, at the rates of 0.15 and 0.30 g a.i./pit and check. Twig samples were collected prior to and 30 , 60, 90, 120, 150, 180, 210 and 240 days after the application, at three coffee plant heights (lower, middle and upper third), and the percentage of mined leaves was evaluated. The determination of aldicarb residues, including their sulphoxide and sulfone metabolites and of thiamethoxam were performed by gas chromatography with a nitrogen-phosphorus and mass spectrometer detectors, respectively. The results indicated a uniform translocation of both insecticides in all three thirds of the coffee plants when applied to the soil. A higher persistence of thiamethoxam was verified with its residues being found for as far long as eight months following the application, while aldicarb residues, including the sulphoxide and sulfone metabolites, were found only until four to six months after the application. Control of the coffee leaf miner was observed with both insecticides.     

The effect of serum,EGF, PGF2α and insulin on S6 phosphorylation and the initiation of protein and DNA synthesis

To test the connection between S6 phosphorylation and the activation of protein and DNA synthesis, we compared the effects of serum, epidermal growth factor (EGF), prostaglandin F_2α (PGF_2α) and insulin (which is not mitogenic in these cells). Increasing concentrations of serum or EGF produced roughly parallel effects on all three processes, though the maximum response elicited by EGF (10^?9 M) was only a portion of that caused by saturating levels of serum (7.5% to 10%). PGF_2α (8.5 × 10^?7 M) alone acted similarly to EGF (10^?9 M) and with EGF produced a synergistic effect on all three processes. Insulin (10^?9 M) alone stimulated both S6 phosphorylation and protein synthesis to approximately the same level as EGF or PGF_2α, but had no effect on initiation of DNA synthesis. Thus neither stimulation of S6 phosphorylation nor activation of protein synthesis is sufficient for initiation of DNA synthesis. The requirement for S6 phosphorylation could not be dissociated from the activation of protein synthesis. Ribosomes containing the most highly phosphorylated forms of S6 appear to have a selective advantage in entering polysomes.     

Semisynthesis of 6-Chloropurine-2′-deoxyriboside 5′-Dimethoxytrityl 3′-(2-Cyanoethyl-N,N-diisopropylamino)Phosphoramidite and its Use in the Synthesis of Fluorescently Labeled Oligonucleotides

An efficient enzymatic synthesis of 6-chloropurine-2′-deoxyriboside from the reaction of 6-chloropurine with 2′-deoxycytidine catalyzed by nucleoside-2′-deoxyribosyltransferase (E.C. 2.4.2.6) followed by chemical conversion into the 5′-dimethoxytrityl 3′-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidite derivative is described. The phosphoramidite derivative was incorporated site-specifically into an oligonucleotide and used for the introduction of a tethered tetramethylrhodamine-cadaverine conjugate. The availability of an efficient route to 6-chloropurine-2′-deoxyriboside 5′-dimethoxytrityl 3′-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite enables the facile synthesis of oligonucleotides containing a range of functional groups tethered to deoxyadenosine residues.     

Identification of the major urinary metabolites of 6-ketoprostaglandin F1α (6K-PGF1α) in the rat

Two main urinary metabolites of 6K-PGF_1α were isolated after intravenous injection of this compound into adult male Wistar rats. The structures were identified as: dinor 6K-PGF_1α and dinor ω-1-hydroxy 6K-PGF_1α. The structures of these two novel products were identified by gas chromatographymass spectrometry of the methyl and ethyl ester derivatives of the O-methyl-oxime trimethylsilyl derivatives and the methyl ester O-methyloxime t-Butyl-dimethylsilyl ether derivatives. These results indicate that the main pathway of metabolism of 6K-PGF_1α$\text{in vivo}$ is via β-and ω-oxidation and not via the prostaglandin 15-hydroxydehydrogenase pathway in this species.     

Comparison of cytokinin activities of the base,ribonucleoside and 5′- and cyclic-3′,5′-monophosphate ribonucleotides of N6-isopentenyl-, N6-benzyl or 8-bromo-adenine

The cytokinin activities of adenosine 3′,5′-monophosphate, N⁶,O^2″-dibutyryladenosine 3′,5−'monophosphate, 8-bromoadenosine 3′,5′-monophosphate, N⁶-(Δ²-isopentenyl)adenosine 3′,5′-monophosphate, and N⁶-benzyladenosine 3′,5′-monophosphate were determined in the tobacco bioassay and compared with the activities of the corresponding non-cyclic nucleotides, nucleosides and bases of the N⁶-isopentenyl-substituted, N⁶-benzyl-substituted, 8-bromo-substituted, and unsubstituted adenine series. In each of these series the cytokinin activities in decreasing order were: bases ⪢ nucleosides ⪖ nucleotides > cyclic nucleotides. All members of the N⁶-isopentenyl- substituted and N⁶-benzyl-substituted series were highly active cytokinins, reaching maximum activity at concentrations of 1 μM or less, whereas, as expected, all members of the unmodified adenine series were inactive in the tested concentration ranges of up to 180 and 200 μM for adenosine and adenine, and 40 μM for the adenine nucleotides. Members of the 8-bromo-substituted adenine series were much weaker cytokinins than the N⁶-substituted adenine derivatives but showed activity in the same sequence starting at a concentration of about 5 μM. Thus, in the cases of 8-bromoadenosine 3′,5′-monophosphate and N⁶,O^2′-dibutyryl-adenosine 3′,5′-monophosphate, both of which have been reported to promote cell division and growth of plant tissues, the cytokinin activity is related to the 8-bromo substituent and to the N⁶-butyryl substituent, respectively, rather than to the 3′,5′-cyclic monophosphate moiety.     

Interaction between CK2α and CK2β, the subunits of protein kinase CK2: thermodynamic contributions of key residues on the CK2α surface

The protein Ser/Thr kinase CK2 (former name: casein kinase II) exists predominantly as a heterotetrameric holoenzyme composed of two catalytic subunits (CK2α) bound to a dimer of noncatalytic subunits (CK2β). We undertook a study to further understand how these subunits interact to form the tetramer. To this end, we used recombinant, C-terminal truncated forms of human CK2 subunits that are able to form the holoenzyme. We analyzed the interaction thermodynamics between the binding of CK2α and CK2β as well as the impact of changes in temperature, pH, and the ionization enthalpy of the buffer using isothermal titration calorimetry (ITC). With structure-guided alanine scanning mutagenesis we truncated individual side chains in the hydrophobic amino acid cluster located within the CK2α interface to identify experimentally the amino acids that dominate affinity. The ITC results indicate that Leu41 or Phe54 single mutations were most disruptive to binding of CK2β. Additionally, these CK2α mutants retained their kinase activity. Furthermore, the substitution of Leu41 in combination with Phe54 showed that the individual mutations were not additive, suggesting that the cooperative action of both residues played a role. Interestingly, the replacement of Ile69, which has a central position in the interaction surface of CK2α, only had modest effects. The differences between Leu41, Phe54, and Ile69 in interaction relevance correlate with solvent accessibility changes during the transition from unbound to CK2β-bound CK2α. Identifying residues on CK2α that play a key role in CK2α/CK2β interactions is important for the future generation of small molecule drug design.     

Influence of IL-6, IL-10, IFN-γ and TNF-α genetic variants on susceptibility to diabetic kidney disease in type 2 diabetes mellitus patients

Background: Data from previous studies on the role of inflammatory cytokines as biomarkers for diabetic kidney disease (DKD) are contradictory. The association of a particular inflammatory cytokine single nucleotide polymorphism (SNP) with susceptibility to DKD has not been consistently replicated. We aimed to investigate the utility of inflammatory cytokines as biomarkers for DKD in type 2 diabetes mellitus (T2DM) patients. Association of inflammatory cytokine gene SNPs with the development of DKD was also explored.

Subjects and Methods: One hundred and fifty-nine Kuwaiti subjects were recruited in this study, including 50 T2DM patients without DKD, 67 diabetic DKD patients and 42 healthy subjects. Plasma levels of interleukin-6 (IL-6), IL-10, interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α) were measured by enzyme-linked immunosorbent assays. Nine SNPs, including 2 SNPs in IL-6, 3 SNPs in IL-10, 1 SNP in IFN-γ and 3 SNPs in TNF-α, were genotyped using TaqMan SNP genotyping assays.

Results: Diabetic DKD patients showed higher IL-6, IL-10, IFN-γ and TNF-α levels than those without DKD. Diabetic DKD patients had a significantly higher frequency of IL-10???1082?A allele than those without DKD (p?=?0.001). No significant association of IL-6???174/?597 haplotypes with DKD risk was detected (p?=?0.188). Distribution of IL-10???592/?819/?1082 haplotypes differ significantly between T2DM patients with/without DKD (p?=?0.014). Diabetic DKD patients had a significantly lower frequency of IL-10???592C/?819C/?1082G haplotype than those without DKD (p?=?0.002).

Conclusions: Although inflammatory cytokine genotypes and, more importantly, haplotypes may have the potential to identify those patients at risk of DKD, hence, improving DKD predisposition prediction, further investigations regarding their real clinical significance is warranted in a large cohort of patients.     

Impact of preconditioning stem cells with all-trans retinoic acid signaling pathway on cisplatin-induced nephrotoxicity by down-regulation of TGFβ1, IL-6, and caspase-3 and up-regulation of HIF1α and VEGF

The survival reduction after transplantation limited the clinical uses of stem cells so the current study explored preconditioning adipose-derived stem cells (ADMSCs) and all-trans retinoic acid (ATRA) effects on cisplatin that caused acute kidney injury (AKI). One hundred and fifty Sprague–Dawley male rats were distributed into five groups: control group; Cisplatin (CIS) group; CIS and ATRA group; CIS and ADMSC group, and CIS, ATRA, and ADMSCs group. Ten rats were euthanized after 3rd, 7th, and 11th days from CIS injection. Renal function, molecular studies, and histopathological analysis were studied. The preconditioning of ADMSCs with ATRA increased the viability of the cells which was reflected in the amelioration of kidney functions after CIS injection by the significant reduction of serum creatinine, microalbuminuria, as well as NO, and the significant rise of creatinine clearance, as well as SOD compared to the group of cisplatin. ATRA also supported ADMSCs by a significant down-regulation of caspase-3, il-6 and TGFβ1, and a significant up-regulation of HIF1, VEGF and CD31 compared to group of cisplatin which reversed the cisplatin effect. ATRA increased renoprotective properties of ADMSCs against cisplatin- induced AKI by reducing the apoptosis, inflammation, and stimulating angiogenesis.     

Isolation and structural analyses of positional isomers of 61,6m-di-O-α-d-mannopyranosyl-cyclomaltooctaose (m=2–5) and 6-O-α-(n-O-α-d-mannopyranosyl)-α-d-mannopyranosyl-cyclomaltooctaose (n=2, 3, 4, and 6)

Eight positional isomers of 6¹,6^m-di-O-α-d-mannopyranosyl-cyclomaltooctaose (γCD) (m=2–5) and 6-O-α-(n-O-α-d-mannopyranosyl)-d-mannopyranosyl-γCD (n=2, 3, 4, and 6) in a mixture of products from γCD and d-mannose by condensation reaction of α-mannosidase from jack bean were isolated by HPLC. The structures of four isomers of 6-O-α-(n-O-α-d-mannopyranosyl)-d-mannopyranosyl-γCD were elucidated by NMR spectroscopy. On the other hand, four positional isomers of 6¹,6^m-di-O-α-d-mannopyranosyl-γCD were determined by LC–MS analysis of degree of polymerization of the branched oligosaccharides produced by enzymatic degradation with bacterial saccharifying α-amylase (BSA), and combination of BSA and glucoamylase. Similarly cyclomaltodextrin glucanotransferase also digested these isomers.     

Effect of growth culture physiological state,metabolites, and formulation on the viability,phytotoxicity, and efficacy of the take-all biocontrol agent Pseudomonas fluorescens 2–79 stored encapsulated on wheat seeds

Strain 2–79 is a biocontrol agent of take-all, an important root disease of wheat caused by Gaeumannomyces graminis var. tritici. In the rhizosphere, strain 2–79 produces the antibiotic phenazine-1-carboxylic acid as the primary means of disease suppression. Barriers to the commercial use of phenazine-producing pseudomonads, such as strain 2–79, include the lack of liquid-culture and formulation technologies needed to optimize cost-effective mass production and application. For instance, there is little published research concerning the impact of growth culture physiological state and associated metabolites on the biocontrol qualities of the cells harvested and formulated in seed coatings, i.e., efficacy, phytotoxicity, and storage survival. To enable exploration of these issues, cells of strain 2–79 in various physiological states were obtained by harvesting fermentors at 24-h intervals after inoculation. Cells formulated in 0.5% methylcellulose suspended in either water (MW) or metabolite-bearing, spent culture broth (MSB) were applied as wheat-seed coatings, air dried, and stored at 4°C. Younger cells (24–48?h) had twice the drying survival rate but only half of the storage life demonstrated by older cells (72–96?h) (P?0.05). Cell populations surviving drying were 3.5 times higher in MW than in MSB formulations and they remained viable up to 3 times longer (P?0.05). This effect of formulation on viability was attributable to the culture nutrients but not the metabolites present in the spent broth. Disease suppression in bacterized seed treatments was significant (P?0.05) relative to unbacterized controls and averaged 9.1%, but did not vary significantly (P?0.24) with culture age, encapsulation medium, or storage time. Relative seedling height improvement increased with relative disease suppression (P=0.003) and significantly decreased with lengthening storage time (P=0.004). This latter decline in plant growth promotion coincided with the deterioration of biocontrol agent viability during storage. Seed batches inoculated with cells in both MW and MSB encapsulations suffered significant germination losses due to phytotoxic metabolites. The extent of loss was an interactive result of encapsulation medium and storage time (P?0.01), and the rate of loss was much higher for seeds with MSB than with MW coatings, i.e. 54% compared to 11% loss after 6 months storage.     

Effect of experimental infection with Listeria monocytogenes on the development of pregnancy and on concentrations of progesterone,oestrone sulphate and 15-ketodihydro-PGF2α in the goat

The effect of Listeria monocytogenes infection on hormone levels in pregnant goats was studied. Four goats (Group I) received an intravenous inoculation of a bacterial culture (Type 1) on Days 69–77 and another four goats (Group II) received a similar inoculation on Days 105–106 of gestation. Five non-inoculated goats were used as controls. Plasma was analysed for progesterone, oestrone sulphate and 15-ketodihydro-PGF_2α. The status of the foetus was followed using real-time ultrasonography.Three of the four goats in Group I aborted 8–10 days after inoculation. The fourth goat gave birth to a normal live kid at term. The three goats which aborted showed clinical signs of disease in connection with abortion. In Group II, all goats aborted after 9–11 days. All the goats showed clinical symptoms of disease from a few days after inoculation and the symptoms continued until abortion. The clinical symptoms of disease were more pronounced in Group II than in Group I. L. monocytogenes was isolated from all aborted foetuses. None of the control goats aborted.Ultrasound examination revealed foetal death either immediately before or up to 2 days before abortion. Mummification had begun in the foetus that had been dead for 2 days before expulsion.In comparison with pre-inoculation plasma levels in Group I, a significant decrease (P < 0.05) in progesterone levels and an increase in 15-ketodihydro-PGF_2α levels were observed from Days 4 and 6 after inoculation, respectively. In Group II, a significant decrease (P < 0.05) in progesterone levels and an increase in 15-ketodihydro-PGF_2α levels in plasma were observed from Days 8 and 6, respectively. The oestrone sulphate levels decreased slightly in the inoculated goats a few days before abortion.The pattern of changes in levels around abortion was similar to the pattern present in the control animals around parturition. However, oestrone sulphate levels did not increase in the inoculated groups before abortion in contrast to goats which delivered healthy kids. The changes in levels of 15-ketodihydro-PGF_2α in goats that aborted indicated that the endocrine foetal-placental function was disturbed, which was most likely due to the establishment and development of L. monocytogenes in the placenta and foetus.     

Binding of STW 5 (Iberogast®) and its components to intestinal 5-HT,muscarinic M3, and opioid receptors

Clinical studies with the fixed herbal combination product STW 5 (Iberogast^®) have indicated an efficacy comparable to metoclopramide (5-HT₃ antagonist) and cisapride (5-HT₄ agonist) in functional gastro-intestinal diseases like functional dyspepsia (FD) and irritable bowel syndrome (IBS). Since serotonin (5-HT₃ and 5-HT₄) and muscarinic M₃ receptors are known to play a central role in the etiology of FD and IBS, the extracts contained in STW 5 and several of their phytochemical components were studied in vitro for binding affinities to these receptors of the intestine. STW 5 inhibited the binding of ³H-GR113808 and ³H-4-DAMP to 5-HT₄ and M₃ receptors, respectively, about 10 times more potently than the binding of ³H-GR65630 to 5-HT₃ receptors. IC₅₀ values for STW 5 did correspond to extract dilutions of 1:1000 (M₃ binding) and 1:2000 (5-HT₄ binding). In addition, STW 5 also potently inhibited the binding to opioid receptors with an IC₅₀ value of 1:2000. Of the nine herbal extracts contained in STW 5, the fresh plant extract of bitter candy tuft (Iberis amara) selectively inhibited binding to M₃ receptors, while ethanolic extracts of celandine herb and chamomile flower were selective to 5-HT₄, and liquorice root to 5-HT₃ receptors. Binding affinities to human recombinant 5-HT₃, 5-HT₄ and M₃ receptors were qualitatively similar to those of the corresponding intestinal receptors. The benzylisoquinoline alkaloid berberine had significant inhibitory action on 5-HT₄ and M₃ binding, showing IC₅₀ values of 40 ng/ml (100 nM) and 200 ng/ml (500 nM), respectively, but is present in the extract of celandine herb only in traces, so that also for the celandine extract a cooperative effect of several phytochemical constituents can be assumed. These in vitro data indicate that 5-HT₄ (to a lesser degree 5-HT₃), muscarinic M₃, and opioid receptors represent target sites for the treatment of FD and IBS with STW 5 (Iberogast^®).