An enzyme-linked immunosorbent assay (ELISA) for the quantitation of urinary desmosine

An enzyme-linked immunosorbent assay (ELISA) method has been developed for the quantitation of the elastin cross-link desmosine in urine. The system employs Rabbit antisera directed to the conjugate of desmosine and bovine serum albumin which was conjugated using carbodiimide reagent. The ELISA was done in microtiter plates which were coated with a desmosine-gelatin conjugate. And the assay system is based on an inhibition immunoassay. With this assay system, desmosine could be detected in a range between 0.4-400 ng/ml. Recovery of desmosine (DES) added to urine was 90.6-117.0% as measured by this method. Anti-DES antisera obtained from rabbits, showed no cross-reaction to 19 standard amino acids, two elastines nor mouse acetone liver power (which contained degradated elastin). But iso-desmosine cross-reacted with the antisera 13-45% at the isodesmosine concentration range of 40-400 ng/ml. Column purification of the urinary desmosine with CF-1 cellulose will not be necessary for desmosine measurement in ELISA assay. This paper described the detail procedures for sample preparation and the desmosine measurement in urine. Desmosine measurement can be an effective marker for screening the lung elastin degradation caused by cigarette smoking and environmental pollution to human lung.     

Photolysis and ozonolysis of (iso) desmosine-containing crosslinked peptides from porcine aorta elastin

This report describes the use of photolysis and ozonolysis as a means of achieving complete cleavage of the pyridinium ring of (iso)desmosine in crosslinked elastin peptides. Although photolysis leads to the opening of the ring with concomitant formation of lysine, the peptide chains remain attached. Subsequent ozonolysis is able to completely achieve the cleavage of the rest of the ring skeleton, thus leading to the separation of the peptide chains. Formation of new amino acids, i.e. α-aminoadipic and glutamic acids, is emphasized. Localization of these amino acids within the released peptides should be of help in structural investigations on the crosslinking zones involving either isodesmosine or desmosine. However, other amino acids such as tyrosine and phenylalanine are sensitive to this procedure and side reactions occur which are responsible for peptide bond cleavage with the formation of breakdown products.     

Characteristic change of urinary elastin peptides and desmosine in the aortic aneurysm.

To estimate elastin metabolism in aneurysm, urinary levels of desmosine and elastin peptide in patients (n=23, range 54 to 85 years old) with aneurysm were measured by ELISA and compared between two control groups divided by age (<10 years old and >20 years old). The amounts of urinary desmosine and elastin peptide in the aneurysm group were significantly increased compared with those in the older control group (>20 years old). There was a correlation between urinary desmosine and elastin peptide in the young group. On the other hand, no such correlation was observed in the aneurysm group and the older control group. The distribution of the ratio (desmosine/elastin peptide) in the aneurysm group was different from that of the young control group. We conclude that assay of elastin peptide and desmosine in urine are useful in characterizing elastin degradation in a patient with aneurysm.     

Experience with an enzyme-linked immuno sorbent assay for the quantitation of urinary desmosine

An enzyme-linked immuno sorbent assay has been set up for the quantitation of the elastin crosslink desmosine. With the assay desmosine could be detected in the range 0.01-10 ng (the amount is corresponding to 0.4-400 ng/ml) in standard solutions. One hundred percent crossreactivity with isodesmosine was found. The low titer rabbit antiserum used in this inhibition immunoassay was less suitable for the measurement of desmosine in urinary hydrolysates, because a number of unknown urinary substances interfered with the measurement. Interfering substances could not be removed completely with a column purification method. Future research is needed to determine the applicability of the higher titer anti-desmosine antiserum from a second rabbit.     

Reversible and irreversible inhibition of rat brain muscarinic receptors is related to different substitutions on bisquaternary pyridinium oximes

The role of the functional substituents on the pyridinium ring of bisquaternary pyridinium compounds, mostly oximes, in exerting reversible and irreversible inhibition of binding of [³H]-N-methyl-4-piperidyl benzilate ([³H]-4NMPB) to rat brain stem muscarinic receptors was studied. The drugs tested, i.e. HGG-42, HGG-12, HGG-52, HI-6, obidoxim, SAD-128 and TMB-4, could reversibly inhibit binding of [³H]-4NMPB, with the highest potency (K_I=1.7–6 M) exhibited by analogs possessing hydrophobic substituents at position 3 or 4 of the pyridinium ring. Bisquaternary drugs possessing an oxime moiety at position 2, but not at position 4 of the pyridinium ring, could also induce about 30% reduction of maximal binding capacity (B_max) (loss of muscarinic receptors) in addition to their reversible effect. Thus the structural correlates of the reversible and the irreversible effects of these drugs are different.     

Modulation by propranolol of the lysyl cross-links in aortic elastin and collagen of the aneurysm-prone turkey

dl-Propranolol (propranolol) fed to immature and mature aneurysm-prone turkeys (Broad-Breasted White, BBW) for 6 weeks significantly raised the tensile strength of tissue rings from the abdominal aorta. The drug-mediated increase in tensile strength values was dose-related and independent of its heart rate- and arterial pressure-lowering effects. Propranolol acts, in part, by (a) stimulating lysyl oxidase to produce greater amounts of reactive aldehydes for intermolecular cross-links, (b) enhancing the progression of chemically unstable to stable forms of intermolecular elastin cross-links (lysinonorleucine and the desmosines), and (c) reducing the density of the age-related intermolecular cross-linking of collagen (pyridinoline). These propranolol effects on the lysyl cross-links were demonstrated in both the immature and mature animals and suggest a heretofore unrecognized potential for this widely used cardiovascular drug.     

Cannabilactones: a novel class of CB2 selective agonists with peripheral analgesic activity

The identification of the CB2 cannabinoid receptor has provided a novel target for the development of therapeutically useful cannabinergic molecules. We have synthesized benzo[ c]chromen-6-one analogs possessing high affinity and selectivity for this receptor. These novel compounds are structurally related to cannabinol (6,6,9-trimethyl-3-pentyl-6 H-benzo[ c]chromen-1-ol), a natural constituent of cannabis with modest CB2 selectivity. Key pharmacophoric features of the new selective agonists include a 3-(1',1'-dimethylheptyl) side chain and a 6-oxo group on the cannabinoid tricyclic structure that characterizes this class of compounds as "cannabilactones." Our results suggest that the six-membered lactone pharmacophore is critical for CB2 receptor selectivity. Optimal receptor subtype selectivity of 490-fold and subnanomolar affinity for the CB2 receptor is exhibited by a 9-hydroxyl analog 5 (AM1714), while the 9-methoxy analog 4b (AM1710) had a 54-fold CB2 selectivity. X-ray crystallography and molecular modeling show the cannabilactones to have a planar ring conformation. In vitro testing revealed that the novel compounds are CB2 agonists, while in vivo testing of cannabilactones 4b and 5 found them to possess potent peripheral analgesic activity.     

Effect of a single injection of cyanamide on the pool of free amino acids in the rat brain

The content of free amino acids in rat brain changes uniformly (serine, taurine, GABA, alanine, valine, cystine, leucin, phenylalanine) as a result of injecting rats with cyanide (60 mg/kg) or cyanide in conjunction with ethanol (0.5 g/kg). In addition, in the latter case the level of cysteic acid and ornithine decreases 2-fold.     

The action of taurine on the response to glutamate in the motoneuron of the isolated frog spinal cord

Using the sucrose gap method, the effect of taurine on the response to glutamate in the ventral root of the isolated frog spinal cord was investigated. The depolarization induced by glutamate was reduced by taurine and the inhibitory action of taurine was antagonized by strychnine, but not by picrotoxin or bicuculline. This action of taurine was also unaffected by tetraethylammonium, 4-aminopyridine and Ringer deficient in sodium ions, but was affected by chloride-free Ringer. Potassium-free Ringer abolished the effect of taurine on the amplitude of the response to glutamate, but not on the rising phase of the depolarization induced by glutamate. Taurine also abolished the after-hyperpolarization induced by glutamate and this was reduced by ouabain or lithium ions. These findings suggest that taurine acts on a glycine receptor to inhibit the response to glutamate, that the action of taurine partly depends on chloride ions, and that taurine inhibits the sodium pump.     

Higher susceptibility of taurine-deficient rats to seizures induced by 4-aminopyridine

The susceptibility of rats made deficient of taurine by treatment with guanidinoethane sulfonate (GES), to seizures induced by 4-aminopyridine was examined. Guanidinoethane sulfonate, at a concentration of 1% was administered to pregnant rats, in the drinking water 2–3 days prior to delivery and the treatment was continued during nursing. Pups were weaned to the same treatment until 6 weeks of age. This treatment decreased levels of taurine in the cerebral cortex by 70%. 4-Aminopyridine was injected intraperitoneally at doses ranging from 4–7 mg/kg. Taurine-deficient rats showed a greater susceptibility to seizures, as demonstrated by a lowered latency for clonic seizures, an increased incidence of tonic seizures and a higher postseizure mortality. These results suggest an involvement of endogenous taurine in nervous excitability.     

Gamma-aminobutyric acid and benzodiazepine receptors in cultured cerebellar granule cells: effects of taurine and its lipophilic derivatives

The effects of taurine and some lipophilic derivatives of taurine on binding to GABA and benzodiazepine receptors were studied in intact cerebellar granule cells. The phenylsuccinylimido derivatives of taurine appeared to increase the binding of muscimol in micromolar concentrations, while taurine decreased it slightly. Only minor changes were seen in the basal binding of flunitrazepam, whereas stimulation of the binding by GABA was strongly reduced by piperidino, benzamido and phenyl-succinylimidotaurine with taurine itself again showing only a weak effect. Diphenylhydantoin, which bears structural resemblance to the phenylsuccinylimido group, had a strong effect on the stimulated binding of flunitrazepam and it also slightly reduced the basal level of binding. Thus, it seems possible that the effects of the phenylsuccinylimido derivatives of taurine on the binding of flunitrazepam were due to this chemical structure and not to the taurine-like core of the molecules. The phthalimido derivative of taurine, taltrimide, which has been tested in clinical trials with epileptic patients, did not show any activity in the binding studies.     

Inhibition by sulfur-containing amino acids and GABA of sympathetic neurotransmission in guinea-pig vas deferens

Electrical stimulation produced a contraction in the isolated guinea-pig vas deferens. This response was blocked by tetrodotoxin, guanethidine and bretylium but not by atropine. The magnitude of the contractile response to electrical stimulation depended on the concentration of the external calcium. Sulfur-containing amino acids and GABA inhibited the electrically induced contraction but not that caused by noradrenaline and ATP. The order of potency for inhibition of the contraction at a concentration of 10(-4) M being GABA greater than or equal to cysteine greater than methionine greater than cysteic acid greater than taurine. The contractile response to electrical stimulation was also inhibited by EGTA, this inhibition being similar to that by cysteic acid and taurine but weaker than that by methionine, cysteine and GABA at a concentration of 10(-4) M. The inhibitory action of sulfur-containing amino acids and GABA was abolished by increasing the calcium concentration in the medium. The results suggest that sulfur-containing amino acids and GABA reduce transmitter release from the sympathetic nerve terminals by inhibiting calcium availability for the transmitter secretion process.     

Influence of muzolimine on arterial wall elastin

Muzolimine, 3-amino-1-(3,4-dichloro-alpha-methylbenzyl)-2- pyrazolin -5-one, an antihypertensive and diuretic drug, accumulates in the arterial tissue of rats and dogs after oral administration. Two weeks after the administration of 3 mg [14C]muzolimine, the aorta of rats contained 60-300 times more 14C-radioactivity/weight unit than the skin or tail tendon. The 14C-radioactivity was exclusively bound to the isolated aortic elastin and corresponded to 0.04% of the applied muzolimine dose. Up to ca 250 ng bound muzolimine/mg elastin was found in the aorta of dogs treated with non-labelled muzolimine for 52 weeks. The elastin-bound [14C]muzolimine was not extractable by organic solvents or by weak acids or bases but was released in a soluble form by pancreatic elastase and extracted from the elastase digest by dichloromethane. In the dichloromethane extract muzolimine was detected by HPLC and HPTLC, and was identified by mass spectrometry. Muzolimine pretreatment of rats for 2 months did not influence the elastin content of arterial tissue or [3H]glycine incorporation into aortic elastin under organ culture conditions, but after labelling the elastin with [4,5-3H]lysine, the [3H]desmosine and [3H]-isodesmosine isolated from the elastin of muzolimine-pretreated rats and incorporated under organ culture conditions was lower than that of control animals. In addition, aortic elastin of rats pretreated for 2 months with 800 ppm muzolimine in the diet was more resistant to elastase degradation. This effect might give some implications for muzolimine in the therapy of cardiovascular disorders with impaired arterial elastin metabolism.     

吸烟诱导慢性阻塞性肺疾病急性加重期血清锁链素水平变化及其与肺功能的相关性研究

目的 探讨吸烟对血清中锁链素(desmosine,DES)表达水平的影响及其与慢性阻塞性肺疾病(COPD)的关系.方法 将COPD急性加重患者分为不吸烟COPD组、吸烟COPD组,同时选择吸烟健康组和不吸烟健康组.收集静脉血,ELISA方法测定血清中的锁链素浓度,对锁链素与吸烟指数、肺通气功能指标FEV1％pred进行直线相关分析.结果 1.吸烟COPD组血清锁链素水平(0.38±0.17) ng/ml高于不吸烟COPD组(0.29±0.08) ng/ml、吸烟健康组(0.21±0.16) ng/ml、不吸烟健康组(0.15±0.07)ng/ml,差异有统计学意义(P＜0.05).2.血清锁链素水平与吸烟指数呈正相关(r=0.412,P＜0.05),与FEV 1％pred呈负相关(r=-0.348,P＜0.05).结论 吸烟COPD患者、不吸烟COPD患者均有使锁链素水平升高的趋势,在吸烟COPD患者中升高更明显.吸烟COPD患者血清锁链素水平与肺功能FEV1％pred下降相关.     

Measurement of cross-reactive properties of adriamycin derivatives by the inhibition enzyme-linked immunosorbent assay for adriamycin

An inhibition enzyme-linked immunosorbent assay (Inhibition ELISA) system for adriamycin has been established. In this assay system, polyclonal anti-adriamycin antiserum produced in rabbits was used. The assay system showed sensitivity for adriamycin in the range of 0.1-2.5 micrograms/ml (0.53-4.23 nmoles per ml). This assay system was applied to analyze the cross-reactive properties of substances with chemical structures similar to that of adriamycin. The cross-reactive properties of six kinds of adriamycin derivatives (Epirubicin, Daunorubicin, Pirarubicin, Aclarubicin, SM-5887 and MX-2) against anti-adriamycin antiserum were measured. The cross-reactive properties of each derivative against the antiserum differed in accordance with the differences in the position of their functional groups attached to the anthracyclic ring. The antiserum recognized the -epi type of -OH group at the -4' position on sugar chains as well as number of sugar chains attached to the anthracyclic ring, and it also recognized the glycoloyl or acetyl and/or ethyl group at position 9 on the anthracyclic ring. With this assay system, the antigenic determinant sites of the anticancer drugs were inferred.     

Quinoline as a privileged scaffold in cancer drug discovery

Quinoline (1-azanaphthalene) is a heterocyclic aromatic nitrogen compound characterized by a double-ring structure that contains a benzene ring fused to pyridine at two adjacent carbon atoms. Quinoline compounds are widely used as "parental" compounds to synthesize molecules with medical benefits, especially with anti-malarial and anti-microbial activities. Certain quinoline-based compounds also show effective anticancer activity. This broad spectrum of biological and biochemical activities has been further facilitated by the synthetic versatility of quinoline, which allows the generation of a large number of structurally diverse derivatives. This includes numerous analogues derived from substitution of the quinoline ring system, and derivatization of quinoline ring structure. Quinoline and its analogs have recently been examined for their modes of function in the inhibition of tyrosine kinases, proteasome, tubulin polymerization and DNA repair. In this review, we have summarized our knowledge on quinoline compounds with respect to their anticancer activities, mechanisms of action, structure-activity relationship (SAR), and selective and specific activity against various cancer drug targets. In particular, we focus our review on in vitro and in vivo anticancer activities of quinoline and its analogs in the context of cancer drug development and refinement.     

SAR index: quantifying the nature of structure-activity relationships

Structure-activity relationships (SARs) can display very different features. Small chemical modifications of active molecules often dramatically alter biological responses. By contrast, structurally diverse molecules can have similar activity. SARs can also be heterogeneous in nature. For example, for structurally diverse molecules with similar activity, closely related analogs might have significant differences in potency. Given the inherent complexity of SARs, it has been very difficult to estimate SAR characteristics from molecular structure. On the basis of systematic correlation of 2D structural similarity and compound potency, we have developed a function termed "SAR Index" that quantitatively describes the nature of SARs and establishes different SAR categories: continuous, discontinuous, heterogeneous-relaxed, and heterogeneous-constrained. These heterogeneous SAR categories are described for the first time. Given a set of active compounds and their potency values, SAR Index calculations can estimate how likely it is to identify structurally distinct molecules having similar activity.     

Dramatically Enhancing the Sensitivity of Immunoassay for Ochratoxin A Detection by Cascade-Amplifying Enzyme Loading

Enzyme-linked immunosorbent assay (ELISA) is widely used in the routine screening of mycotoxin contamination in various agricultural and food products. Herein, a cascade-amplifying system was introduced to dramatically promote the sensitivity of an immunoassay for ochratoxin A (OTA) detection. Specifically, a biotinylated M13 bacteriophage was introduced as a biofunctional competing antigen, in which a seven-peptide OTA mimotope fused on the p3 protein of M13 was used to specifically recognize an anti-OTA monoclonal antibody, and the biotin molecules modified on capsid p8 proteins were used in loading numerous streptavidin-labeled polymeric horseradish peroxidases (HRPs). Owing to the abundance of biotinylated p8 proteins in M13 and the high molar ratio between HRP and streptavidin in streptavidin-polyHRP, the loading amount of HRP enzymes on the M13 bacteriophage were greatly boosted. Hence, the proposed method exhibited high sensitivity, with a limit of detection of 2.0 pg/mL for OTA detection, which was 250-fold lower than that of conventional ELISA. In addition, the proposed method showed a slight cross-reaction of 2.3% to OTB, a negligible cross-reaction for other common mycotoxins, and an acceptable accuracy for OTA quantitative detection in real corn samples. The practicability of the method was further confirmed with a traditional HRP-based ELISA method. In conclusion, the biotinylated bacteriophage and polyHRP structure showed potential as a cascade-amplifying enzyme loading system for ultra-trace OTA detemination, and its application can be extended to the detection of other analytes by altering specific mimic peptide sequences.     

Monoclonal antibodies to the nonpeptide angiotensin II receptor antagonist, losartan.

Two murine monoclonal antibodies were produced to losartan (DuP 753), a nonpeptide angiotensin II receptor antagonist. Using a solid phase competitive enzyme-linked immunosorbent assay (ELISA), each antibody was examined for its ability to bind to a set of losartan analogs that differ structurally in varying degrees. Both antibodies distinguished fine structural changes in the analogs, particularly at the R5 position of the imidazole ring. No cross-reactivity towards either antibody was observed with the natural ligand angiotensin II, the peptide antagonist saralasin, or the AT2 selective nonpeptide antagonist PD123177.     

Design,synthesis, and structure-activity relationship studies of novel 6,7-locked-[7-(2-alkoxy-3,5-dialkylbenzene)-3-methylocta]-2,4,6-trienoic acids

Retinoid X receptor:peroxisome proliferative-activated receptor (RXR:PPAR) heterodimers play a critical role in the regulation of glucose (RXR/PPARgamma) and lipid metabolism (RXR/PPARalpha). Previously, we described a concise structure-activity relationship study of selective RXR modulators possessing a (2E,4E,6Z)-3-methyl-7-(3,5-dialkyl-6-alkoxyphenyl)-octa-2,4,6-trienoic acid scaffold. These studies were focused on the 2-position alkoxy side chain. We describe here the design and synthesis of a novel series of RXR selective modulators possessing the same aromatic core structure with the addition of a ring locked 6-7-Z-olefin on the trienoic acid moiety. The synthesis and structure-activity relationship studies of these 6,7-locked cyclopentenyl, phenyl, thienyl, furan, and pyridine-trienoic acid derivatives is presented herein.