Poly(U)-directed peptide-bond formation from the 2′(3′)-glycyl esters of adenosine derivatives

Summary The self-condensation of 2(3)-O-glycyl esters of adenosine, adenosine-5-(O-methylphosphate) and P₁, P₂-diadenosine-5-pyrophosphate in 6.2 mM solutions at pH 8.0 and -5°C in the presence of 12.5 mM poly(U) yields approximately 3 times as much diketopiperazine as reactions without poly(U). As the concentration of 2(3)-O-(glycyl)-P₁, P₂-diadenosine-5-pyrophosphate is decreased from 6.2 mM to 1.5 mM the yield of diketopiperazine in the presence of poly(U) decreases slightly from 6.6% to 5.2%, whereas, in the absence of poly(U) the yield of diketopiperazine decreases substantially from 2.4% to 0.75%. The enhanced yield of diketopiperazine that is attributed to the template action of poly(U) is temperature dependent and is observed only at temperatures below 10°C (5°C to -5°C) for 6.2 mM 2(3)-O-(glycyl)-adenosine-5-(O-methylphosphate) and below 23°C (15°C to -5°C) for 6.2 mM 2(3)-O-(glycyl)-P₁, P₂-diadenosine-5-pyrophosphate. The absence of a template effect at high temperatures is attributed to the melting of the organized helices. The hydrolysis half-lives at pH 8.0 and -5°C of 2(3)-O-(glycyl)-adenosine, 2(3)-O-(glycyl)-adenosine-5-(O-methylphosphate), 2(3)-O-(glycyl)-P₁, P₂-diadenosine-5-pyrophosphate, and 5-O-(glycyl)-adenosine in the presence of poly(U) are substantially larger than their half-lives in the absence of poly(U). The condensation of 2(3)-O-(glycyl)-adenosine yields 5% of 5-O-(glycyl)-adenosine in the presence of poly(U) compared to 0.7% in the absence of poly(U).Abbreviations DKP diketopiperazine - (gly)₂ glycylglycine - (gly)₃ glycylglycylglycine - AppA-gly 2(3)-O-(glycyl)-P₁, P₂-diadenosine-5-pyrophosphate - MepA-gly 2(3)-O-(glycyl)-adenosine-5-(O-methylphosphate) - Ado-2(3)-gly 2(3)-O-(glycyl)-adenosine - Ado-5-gly 5-O-(glycyl)-adenosine - Boc-gly N-tert-butyloxycarbonylglycine - AppA P₁, P₂-diadenosine-5-pyrophosphate - MepA adenosine-5-(O-methylphosphate) - AppA-Boc-gly 2(3)-O-(Boc-glycyl)-P₁, P₂-diadenosine-5-pyrophosphate - Ado-5-Boc-gly 5-O-(Boc-glycyl)-adenosine - Ado-2(3)-Boc-gly 2(3)-O-(Boc-glycyl)-adenosine     

The effect of apolipoprotein E polymorphism on plasma cholesteryl ester transfer protein activity in type 2 diabetic patients

We studied the relationship between apo E polymorphism and cholesteryl ester transfer protein (CETP) activity in 127 type 2 diabetic patients who did not take lipid lowering drugs. Furthermore, we studied the relationship between apo E and cholesteryl ester transfer protein (CETP) in modulating plasma triglyceride and HDLcholesterol. Apo E genotypes were determined by PCR-RFLP, and CETP activity was measured using an exogenous way. Our results showed that the CETP activity increased significantly in the E2 carrier group compared to E4 carriers and E3/E3 homozygous (84.7 ± 43.9 vs. 62.5 ± 35.9 vs. 52.6 ± 23.6 nmol CE/ml/2h, respectively; p = 0.015). However, there was no association between apo E polymorphism and lipid parameter variations. Even after adjustment for CETP activity, the results remained unchanged, showing that CETP did not step in the relationship between apo E and lipid parameter variations. In conclusion there is an association between apo E polymorphism and CETP activity, and this association did not affect the relationship between apo E polymorphism and triglyceride and HDLcholesterol concentrations. Published in Russian in Molekulyarnaya Biologiya, 2008, Vol. 42, No. 6, pp. 931–935. The text was submitted by the authors in English.     

Heparin localization and fine structure regulate Burkitt's lymphoma growth

Burkitt's lymphoma (BL) is a B-cell malignancy associated with the Epstein-Barr virus (EBV). Mounting evidence has implicated heparan sulfate proteoglycans and heparan sulfate-like glycosaminoglycans (HSGAGs) in the initiation, severity, and progression of the malignancy. The importance of HSGAGs in regulating BL cell growth was therefore examined. Extracellular exogenous heparin inhibited cell growth >30%, while heparin internalized with poly(beta-amino ester)s promoted proliferation up to 58%. The growth-modulating effects of heparin and internalized heparin were dependent on cell surface HSGAGs, PI3K, and Erk/Mek. Treatment of cells with protamine sulfate or with heparinases potently inhibited proliferation, with the greatest effects induced by heparinase I. Cell surface HSGAGs therefore play an important role in regulating BL proliferation and may offer a potential target for therapeutic intervention.     

A study of triflupromazine in dominant-lethal, cytogenetic and host-mediated assays

The phenothiazine psychotherapeutant, triflupromazine (TFP), was studied for mutagenic potential in dominant-lethal, in vivo and in vitro cytogenetic and host-mediated assay procedures. No evidence of gross chromosomal aberrations or point mutations was detected in these assays even at dosage regimens which produced substantial lethality. The effect of the drug on body temperature was measured at the same doses used for mutagenicity testing. A marked and sustained temperature reduction occurs shortly after administration of as little as 10 mg/kg. Due to the pronounced physiological effects at these levels, the validity of mutagenicity studies conducted at the same levels may be seriously questioned.     

The WD repeat protein FAN regulates lysosome size independent from abnormal downregulation/membrane recruitment of protein kinase C

FAN (factor associated with neutral sphingomyelinase [N-SMase] activation) exhibits striking structural homologies to Lyst (lysosomal trafficking regulator), a BEACH protein whose inactivation causes formation of giant lysosomes/Chediak-Higashi syndrome. Here, we show that cells lacking FAN show a statistically significant increase in lysosome size (although less pronounced as Lyst), pointing to previously unrecognized functions of FAN in regulation of the lysosomal compartment. Since FAN regulates activation of N-SMase in complex with receptor for activated C-kinase (RACK)1, a scaffolding protein that recruits and stabilizes activated protein kinase C (PKC) isotypes at cellular membranes, and since an abnormal (calpain-mediated) downregulation/membrane recruitment of PKC has been linked to the defects observed in Lyst-deficient cells, we assessed whether PKC is also of relevance in FAN signaling. Our results demonstrate that activation of PKC is not required for regulation of N-SMase by FAN/RACK1. Conversely, activation of PKC and recruitment/stabilization by RACK1 occurs uniformly in the presence or absence of FAN (and equally, Lyst). Furthermore, regulation of lysosome size by FAN is not coupled to an abnormal downregulation/membrane recruitment of PKC by calpain. Identical results were obtained for Lyst, questioning the previously reported relevance of PKC for formation of giant lysosomes and in Chediak-Higashi syndrome. In summary, FAN mediates activation of N-SMase as well as regulation of lysosome size by signaling pathways that operate independent from activation/membrane recruitment of PKC.     

Induced reversion of a Chinese hamster ovary triple auxotroph. Validation of the system with several mutagens

A Chinese hamster ovary triple auxotroph (CHO AUXB1) requires glycine, adenosine, and thymidine (GAT) for growth and survival due to a defect in the structural gene for folylpolyglutamate synthetase (FPGS). This auxotroph and others like it contain less than 3% of the parental amounts of FPGS activity. In order to develop a reverse mutation assay with CHO AUXB1, we determined the optimal conditions for measuring reversion and characterized some of the revertants. We also obtained quantitative mutagenicity data for several direct-acting mutagens for comparison to the parental CHO-S/HGPRT locus. Induced revertants appear in the culture immediately following 20-22 h exposures in +GAT complete medium, indicative of dominant genetic changes. They are maximally expressed after 2 population doublings and can be conveniently selected after 44-48 h of expression growth by plating 1 X 10(6) cells/100-mm dish into -GAT-deficient medium and incubating 12-13 days. Plating reconstruction experiments show that the cloning efficiencies of revertants in -GAT medium are not influenced by the presence of up to 1 X 10(6) CHO AUXB1 cells. Dose-dependent increases above the spontaneous revertant frequency (average = 5 X 10(7)) are induced with cis-Pt(NH3)2Cl2 (14-fold) (but not trans-Pt(NH3)2Cl2), PtCl4(10-fold), Pt(SO4)2 (14-fold), K2CrO4 (8-fold), EMS (10-fold), 4-NQO (53-fold), ICR-191 (60-fold), and ICR-170 (30-fold). All of the revertants that have been isolated are stable to repeated subculturing in -GAT medium; 40 out of 42 that have been analyzed are characterized by an increased 72-h growth incorporation of labeled folate and their extracts contain 5-94% as much FPGS as the original, parental CHO-S line. Spontaneous and induced reversion to the GAT+ phenotype primarily reflects mutations involving the FPGS gene locus. But the re-acquisition by most of the revertants of much less than normal amounts of FPGS activity suggests that they arise from compensatory second-site mutations within this gene. Comparison of the mutagenicity patterns of the foregoing compounds as a function of the applied concentration and the relative percent survival reveals some interesting similarities, as well as differences, between the CHO AUXB1/FPGS and CHO-S/HGPRT loci. In particular, the FPGS locus is rather insensitive to EMS (or other simple alkylating agents). However, it seems to be quite susceptible to reversion by other chemicals that are known to react selectively with guanine bases in DNA. CHO AUXBI is a useful supplemental mammalian assay system for assessing quantitatively the generally weak mutagenic activities of metal compounds.     

Antibacterial Flavonoids and Other Compounds from the Aerial Parts of Vernonia guineensis Benth. (Asteraceae)

An extensive phytochemical study of the aerial parts of Vernonia guineensis Benth. (Asteraceae) led to the isolation of a new flavone, vernoguinoflavone and a naturally isolated glycerol ester, eicosanoic acid 2‐hydroxy‐1,3‐propanediyl ester, together with eighteen known secondary metabolites including quercetin, luteolin, vernopicrin, vernomelitensin, β‐amyrin, oleanolic acid, ursolic acid, lupeol, betulinic acid, β‐carotene, a mixture of stigmasterol and β‐sitosterol, β‐sitosterol‐3‐O‐β‐D‐glucoside, 2,3‐dihydroxypropyl heptacosanoate, pentacosanoic acid, docosan‐1‐ol, tritriacontan‐1‐ol, and heptatriacontan‐1‐ol. Eleven compounds are reported herein for the first time from this species. The structures of these compounds were elucidated on the basis of extensive spectroscopic analyses, particularly 1D and 2D NMR, and HR‐ESI‐MS and by comparison of their data with those reported in the literature. The crude extract, fractions and some isolated compounds were evaluated for their antibacterial activity against Gram‐negative bacteria: Escherichia coli (ATCC 25922), Shigella flexineri (NR 518), Salmonella muenchen, Salmonella typhimurium and Salmonella typhi (ATCC 19430). All the tested compounds demonstrated inhibitory activities against the tested enteric bacteria with MIC values ranging from 3.12 to 100 μg/ml. Three flavonoids isolated from the most active fraction demonstrated the best bioactivities against Escherichia coli, Salmonella muenchen and Salmonella typhimurium with MIC values ranging from 3.12 to 25 μg/mL.     

Protective effects of luteolin-7-O-β-D-glucuronide methyl ester from the ethyl acetate fraction of Lycopi Herba against pro-oxidant reactive species and low-density lipoprotein peroxidation

In this study the potent scavenging activity of “Lycopi Herba” (LH) extract was studied using the following: evaluation of the total phenolics, measuring the antioxidant activity by Trolox equivalent antioxidant concentration, measuring the scavenging effects on reactive oxygen species, on reactive nitrogen species, and measuring the inhibitory effect on Cu²⁺ induced human low-density lipoprotein oxidation in vitro. The ethyl acetate fraction from the LH extracts were found to have a potent scavenging activity against all of the reactive species tested, as well as an inhibitory effect on LDL oxidation. Therefore, we isolated and identified luteolin-7-O-β-D-glucuronide methyl ester as the major compound from the ethyl acetate fraction of LH and their antioxidant activities were evaluated.