A semiautomated electron capture gas chromatographic assay of 3-methoxy-4-hydroxyphenylethyleneglycol in urine

A semiautomated method for the assay of 3-methoxy-4-hydroxyphenylethyleneglycol (MHPG) in urine has been developed. The method incorporates a new efficient (95%) extraction procedure combined with an automated gas chromatographic system. This system (consisting of a pulsed electron capture detector and an automatic sample injector which valves the sample between two columns) will accept continuous, unattended, sequential sample input. The limit of sensitivity of the trifluoroacetic anhydride derivative of pure MHPG is 3 pg. Analysis of samples from the same 24-h urine (n = 21) over a 2-month period resulted in a mean of 794 ng/ml with a coefficient of variation of 3.5%. MHPG levels from 12 control males and 9 females with no history of psychiatric disorder, were found to contain 1605 and 1034 μg/24 h, respectively.     

Hydrolysis of diepptide 2-naphthylamides by human muscle enzymes.

Human muscle enzymes that hydrolyse dipeptide 2-naphthylamides were separated by chromatography on DEAE-Sephadex. Evidence was obtained that these substrates are hydrolysed by arylamidase as well as by dipeptidyl aminopeptidases.     

Electrochemical assay of plasmin activity and its kinetic analysis

A sensitive and convenient electrochemical assay of plasmin activity and its kinetic analysis are described. Thus, a ferrocenyl peptide substrate (FcPS) having a plasmin-specific substrate sequence, Lys-Thr-Phe-Lys, and a Cys residue was prepared and immobilized on a gold electrode through the sulfur-gold linkage. The obtained electrode showed a redox signal based on the ferrocene moiety, suggesting the immobilization of FcPS on the electrode. After treatment of this electrode with plasmin, its electrochemical signal was decreased in proportion to an increase of the amount of plasmin. The detection limit for plasmin in this assay system was as low as 50 ng/ml or 0.15 mU/ml. Real-time monitoring of plasmin reaction on the electrode could also be achieved, and the kinetic parameters of this enzymatic reaction could be determined; for example, the k_cat/K_m value was 0.063 μM⁻¹ s⁻¹. Furthermore, a quantitative assay for streptokinase as a plasminogen activator was also demonstrated by using this system.     

Fluorescence polarization assay of plasmin, plasminogen, and plasminogen activator

A chromatographic method involving medium-pressure liquid chromatography on alumina impregnated with silver nitrate is described for the separation of a series of closely related C₂₇ sterol precursors of cholesterol differing only in the number and location of olefinic double bonds. The features of the described system are compared with those of previously described thin-layer, gas-liquid, gravity column, and high-pressure liquid chromatographic methods.     

Conversion of low-affinity platelet factor 4 to beta-thromboglobulin by plasmin and trypsin

Low-affinity platelet factor 4 and beta-thromboglobulin are platelet-secreted proteins that bind with low affinity to heparin. They show extensive immunological cross-reactivity and appear to differ in amino acid sequence only by an amino-terminal peptide unique to low-affinity platelet factor 4. The possibility that beta-thromboglobulin is derived from low-affinity platelet factor 4 by proteolysis was investigated by exposing this protein to the action of plasmin, thrombin and trypsin. While thrombin had no effect, plasmin and trypsin converted low-affinity platelet factor 4 to a species with the same electrophoretic mobility and isoelectric point as beta-thromboglobulin. We conclude that beta-thromboglobulin is a breakdown product of low-affinity platelet factor 4.     

Potentiation of TLR4 signalling by plasmin activity

The potential for proteases to regulate mammalian TLR signalling is controversial. We found that inhibition of extracellular serine proteases did not reduce activation of TLR4, but observed that the protease plasmin, an important fibrinolytic plasma enzyme that also exerts proinflammatory functions in monocytes, potentiated TLR2 and TLR4 signalling in RAW264.7 macrophages. Plasmin enhanced endogenous production of TNFalpha and activation of an NF-kappaB reporter plasmid. These actions were prevented by inhibition of its proteolytic activity and were not recapitulated by agonists of protease-activated receptors. These studies link fibrinolysis and TLR signalling, identifying further mechanisms potentially involved in activation of innate immunity.     

A sensitive fluorometric assay for plasminogen, plasmin, and streptokinase

A rapid, convenient, and highly sensitive fluorometric assay for plasmin (P), plasminogen (Pg), and streptokinase (SK) as the activator complex (SK·P) is described. These assays are based on the measurement of the fluorescence of β-naphthol (βN) released from α-N-methyl α-N-tosyl-l-lysine β-naphthol ester (MTLNE) by the P present or generated during the assay. The rate of βN release may be followed by direct recording or determined subsequently, following termination of the enzyme reaction at a fixed digestion time, by the addition of p-nitrophenyl-p′-guanidinobenzoate. The latter method may be readily automated. The K_m and V values for the hydrolysis of MTLNE by P or SK·P are equivalent. The Pg activator activity of P was shown to be very small (less than 0.2% of that of SK·P).     

Synthesis and antiproliferative evaluation of certain 4-anilino-8-methoxy-2-phenylquinoline and 4-anilino-8-hydroxy-2-phenylquinoline derivatives

The present report describes the synthesis and antiproliferative evaluation of certain 4-anilino-8-methoxy-2-phenylquinoline and 4-anilino-8-hydroxy-2-phenylquinoline derivatives. The antiproliferative activity of 4'-COMe-substituted derivatives decreased in an order of 6-OMe (1, 3.89 microM) > 8-OMe (8, 10.47 microM) > 8-OH (9, 14.45 microM), indicating that the position of substitution at the quinoline ring is crucial. For 3'-COMe derivatives, the antiproliferative activity of 8-OH (11, 1.20 microM) is more potent than its 8-OMe counterpart (10, 8.91 microM), indicating that a H-bonding donating substituent is more favorable than that of a H-bonding accepting group. Comparison of 8-OH derivatives, the antiproliferative effect of COMe (11) is more potent than its oxime derivative (15a, 2.88 microM), which in turn is more potent than the methyloxime counterpart (15b, 5.50 microM). Compound 11 is especially active against the growth of certain solid cancer cells such as HCT-116 (colon cancer), MCF7, and MDA-MB-435 (breast cancer) with GI50 values of 0.07, <0.01, and <0.01 microM, respectively. Flow cytometric analyses revealed that growth inhibition by 11 and 15a was due to accumulation in S-phase. This result is interesting because 2-phenylquinolone derivatives have been reported to be antimitotic agents which induced cell cycle arrest in G2/M phase.     

Conversion of low-affinity platelet factor 4 to β-thromboglobulin by plasmin and trypsin

Low-affinity platelet factor 4 and β-thromboglobulin are platelet-secreted proteins that bind with low affinity to heparin. They show extensive immunological cross-reactivity and appear to differ in amino acid sequence only by an amino-terminal peptide unique to low-affinity platelet factor 4. The possibility that β-thromboglobulin is derived from low-affinity platelet factor 4 by proteolysis was investigated by exposing this protein to the action of plasmin, thrombin and trypsin. While thrombin had no effect, plasmin and trypsin converted low-affinity platelet factor 4 to a species with the same electrophoretic mobility and isoelectric point as β-thromboglobulin. We conclude that β-thromboglobulin is a breakdown product of low-affinity platelet factor 4.     

Fluorescent assay for riboflavin binding to cytochrome P450 2B4

The interactions between the hemoprotein cytochrome P450 2B4 (CYP 2B4) and riboflavin - a low molecular weight component of the flavoprotein NADPH-dependent cytochrome P450 reductase - were investigated by fluorescence spectroscopy. Riboflavin fluorescence quenching by cytochrome P450 2B4 was used to probe the ligand-enzyme binding (lambda(ex)=385 nm, lambda(em)=520 nm). Fluorescence titration experiments showed formation of a complex between cytochrome P450 2B4 and riboflavin with an apparent dissociation constant value, K(d)=8.8+/-1 microM. The fluorescence intensity of riboflavin was decreased with increasing the cytochrome P450 2B4 concentration, indicating the transfer of resonance excitation energy from riboflavin (energy donor) to the cytochrome P450 2B4 heme (energy acceptor). The data obtained are suggestive of the existence of riboflavin binding site(s) on the hemeprotein molecule.     

On the mode of biodegradation of cinnamic acids by Colletotrichum gloeosporoides: A direct access to 4-vinylphenol and 2-methoxy-4-vinylphenol

The mode of transformation by growing cultures of Colletotrichum gloeosporoides of C-6-C-3, unsaturated cinnamic acids 1, a-f depends upon the pattern of the oxygen substitution in the aromatic ring; from 1b and 1d are exclusively obtained upon decarboxylation the C-6-C-2 vinyl phenols 2b and 2d. whereas in the remaining instances the C-6-C-3 educts 3–5 formed by reduction are prevalent     

Use of a computer to assay motility in bacteria

An assay was developed to study the movement of free-swimming Escherichia coli. Cells were videotaped through a microscope, and the videotape images were then digitized and analyzed with a computer. Angular and linear speeds were measured for wild-type E. coli and for a smooth and a tumbly mutant. The average angular and linear speeds of a population were directly and inversely proportional, respectively, to the time spent tumbling. Changes in angular and linear speeds were followed during the response of wild-type E. coli to attractant or repellent.     

The role of a contaminant in thrombin in the human plasmin assay system

Many of the anomalous results obtained in the fibrinolytic assay of human plasmin systems were shown to be simply explained if bovine plasminogen had been introduced into the assay system on the addition of thrombin. Experimental investigation of the proteolytic and fibrinolytic activity of systems containing plasmin and thrombin showed that enzyme activity was influenced by the presence and quantity of thrombin. The quantity of bovine plasminogen present as a contaminant in bovine fibrinogen was shown to be responsible for only 1/25th of the observed enhanced activity. Thrombin in the amounts commonly used for clotting contained sufficient proenzyme to account for all this activity. A highly purified thrombin preparation obtained from another laboratory, and thrombin purified in this laboratory by starch electrophoresis brought about no enhancement of activity. The material separated from thrombin by starch electrophoresis was shown to be enzymatically identical with bovine plasminogen and, on labelling with radioactive iodine, was shown to behave physically like bovine plasminogen. Several experiments reported in the literature were reinterpreted in the light of this observation.     

Suzuki cross-coupling approaches to the synthesis of bioactive 3-substituted and 5-substituted-4-methoxy-6-methyl-2-pyrones

Suzuki cross-coupling has been used to access a wide range of 3- and 5-substituted 2-pyrones, which show remarkable inhibitory activity against bacteria, yeasts and fungi. 3-Octenyl and 5-octenyl 2-pyrones inhibit human ovarium carcinoma (A2780) and human chronic myelogenous leukaemia (K562) cell lines at the micromolar level.     

Combination of 2-methoxy-3-phenylsulfonylaminobenzamide and 2-aminobenzothiazole to discover novel anticancer agents

The fragment of 2-substituted-3-sulfonylaminobenzamide has been proposed to replace the fragment of 2-substituted-3-sulfonylaminopyridine in PI3K and mTOR dual inhibitors to design novel anticancer agents based on bioisostere. The combination of the fragment of 2-substituted-3-sulfonylaminobenzamide with the fragment of 2-aminobenzothiazole or 2-aminothiazolo[5,4-b]pyridine, or 2-amino[1,2,4]triazolo[1,5-a]pyridine produced the novel structures of anticancer agents. As a result, nineteen target compounds were synthesized and characterized. Their antiproliferative activities in vitro were evaluated via MTT assay against four human cancer cell lines including HCT-116, A549, MCF-7 and U-87 MG. The SAR of target compounds was preliminarily discussed. Compound 1g with potent antiproliferative activity was examined for its effect on the AKT and p-AKT⁴⁷³. The anticancer effect of 1g was evaluated in established nude mice HCT-116 xenograft model. The results suggested that compound 1g can block PI3K/AKT/mTOR pathway and significantly inhibit tumor growth. These findings strongly support our assumption that the fragment of benzamide can replace the pyridine ring in some PI3K and mTOR dual inhibitor to design novel anticancer agents.     

The flavonoids of Megalodonta beckii and isolation of 2′-3-Dihydroxy-4-methoxy-4′-glucosyl chalcone

A new naturally occurring chalcone glycoside, $\text{2′,-}\text{dihydroxy}\text{-4-}\text{methoxy}\text{-4′-O-β-}\text{d}\text{-}\text{glucosyl}$ chalcone, and other chalcones, aurones, flavonols, and anthocyanins are reported from Megalodonta (Bidens) beckii. The plan is an amphibious aquatic with heterophyllous leaves, but the presence of certain flavonoids is correlated with vegetative versus floral tissues rather than vegetative leaf form. The implications of flavonoid chemistry with respect to the relationship of M. beckii to several sections of Bidens are discussed.