Direct radioimmunoassay of progesterone in milk using a radioiodinated progesterone derivative

A heterologous radioimmunoassay technique which allows the measurement of progesterone in whole milk, without extraction is described. An antiserum against progesterone-12α-succinyl-BSA and progesterone-11α-(2′-methyl)succinyl-¹²⁵I-thyramine was used. When applied in pregnancy detection on day 20–22, progesterone levels of up to 6 ng/ml or above 8 ng/ml were indicative of an unsuccessful or a successful insemination, respectively.The sensitivity of the assay was 0.8 ng/ml, and the values found in cow milk ranged from 0.8 to 50 ng/ml.     

Synthesis and biologic evaluation of a radioiodinated quinazolinone derivative for enzyme-mediated insolubilization therapy

We have developed a new strategy that aims to concentrate therapeutic radionuclides within solid tumors. This approach, which we have named EMIT (enzyme-mediated insolubilization therapy), is a method for enzyme-dependent, site-specific, in vivo precipitation of a radioactive molecule (from a water-soluble precursor) within the extracellular space of solid tumors. The prodrug, ammonium 2-(2'-phosphoryloxyphenyl)-6-iodo-4-(3H)-quinazolinone, labeled with iodine-125 ((125)IPD) and its authentic compound labeled with iodine-127 (IPD) have been synthesized, purified, and characterized. The alkaline phosphatase (ALP)-mediated conversion of these water-soluble nonfluorescent prodrugs to the water-insoluble fluorescent species, iodine-125-labeled 2-(2'-hydroxyphenyl)-6-iodo-4-(3H)-quinazolinone ((125)ID) and its iodine-127-labeled derivative (ID), has been demonstrated in vitro. Biodistribution studies in mice indicate that both (125)IPD and (125)ID are minimally retained by most tissues and organs. In addition, following its intravenous injection in mice, (125)IPD is localized in ALP-rich regions and converted to (125)ID, which remains indefinitely within the tissues where it is produced. We believe that EMIT is a strategy that will lead to the active and specific concentration and entrapment of therapeutic radionuclides within solid tumors, the consequent protracted irradiation of tumor cells within the range of the emitted particles, and the effective therapy of solid tumors.     

Evaluation of chemical, physical, and biologic properties of tumor-targeting radioiodinated quinazolinone derivative

Our group is developing a novel technology, enzyme-mediated cancer imaging and therapy (EMCIT), that aims to entrap radioiodinated compounds within solid tumors for noninvasive tumor detection and therapy. In this approach, a water-soluble, radioiodinated prodrug is hydrolyzed in vivo to a highly water-insoluble compound by an enzyme overexpressed extracellularly by tumor cells. We have synthesized and characterized the water-soluble prodrug, 2-(2'-phosphoryloxyphenyl)-6-[(125)I]iodo-4-(3H)-quinazolinone [(125)I]5, which is readily hydrolyzed by alkaline phosphatase, an enzyme expressed by many tumor cell lines, to a water-insoluble drug, 2-(2'-hydroxyphenyl)-6-[(125)I]iodo-4-(3H)-quinazolinone [(125)I]1. In the course of our study, we discovered that ammonium 2-(2'-phosphoryloxyphenyl)-6-tributylstannyl-4-(3H)-quinazolinone, an intermediate in the radioiodination of the prodrug, exists as two isomers (3 and 4) whose radioiodination leads, respectively, to [(125)I]6 and [(125)I]5. These prodrugs have different in vitro and in vivo biologic activities. Compound 6 is not hydrolyzed by alkaline phosphatase (ALP), whereas 5 is highly soluble (mg/mL) in aqueous solution and is rapidly dephosphorylated in the presence of ALP to 1, a water-insoluble molecule (ng/mL). Mouse biodistribution studies indicate that [(125)I]6 has high uptake in kidney and liver and [(125)I]5 has very low uptake in all normal organs. Compounds 3 and 6 are converted, respectively, to 4 and 5 after incubation in DMSO. The stability of 5 in human serum is high. The minimum ALP concentration needed to hydrolyze 5 is much greater than the ALP level in the blood of patients with cancer, and the latter should not affect the pharmacokinetics of the compound. Incubation of 5 with viable human and mouse tumor-cell lines--but not with normal human cells and mouse tissues--leads to its hydrolysis and the formation of large crystals of 1. We expect that 5 will also be hydrolyzed in vivo by tumor cells that express phosphatase activity extracellularly and anticipate the specific precipitation of radioiodinated 1 within tumor cell clusters. This should lead to high tumor-to-normal-tissue ratios and enable imaging (SPECT/PET) and radionuclide therapy of solid tumors.     

Pepsin inhibition by a high specific activity radioiodinated derivative of pepstatin.

Stop-flow kinetic studies are reported for the reaction of glutathione (GSH) with 2-chloromercuri-4-nitrophenol. The second-order rate constant was found to vary by less than twofold in going from pH 5.9 to 7.3, suggesting that the reaction involves attack of the un-ionized glutathione thiol on the mercurial around pH 7. The rate constant was 1.5 × 10⁷m^?1 s^?1 at pH 7.0 and 8 °C in dilute buffer of ionic strength 0.2. The sensitivity of the kinetics to the nature of the leaving group in the mercurial i.e., to the labile ligand, was investigated in complexes of the mercurial with OH^?, N₃^?, Br^?, Cl^?, CN^?, SCN^?, pyrophosphate, ADP, ATP, EDTA, and adenosine. The addition of most of these substances was observed to lead to rate enhancements or rate retardations, the kinetic effects showing typical binding isotherm behavior when plotted as a function of the added ligand concentration. The kinetic midpoints of such plots showed very good correlation with the independently determined affinities they had toward the mercurial. The results demonstrate that many commonly added compounds of biochemical interest can significantly influence mercurial reactivity toward thiols through rapid exchange at the labile ligand position of the mercurial.     

Synthesis and biological evaluation of radioiodinated 2NUBTA as a cerebral ischemia marker

N-[4-(Benzothiazol-2-yl)phenyl]-11-(2-nitroimidazole-1-yl)undecanamide (2NUBTA) was synthesized and radiolabeled with iodine-131. In vitro evaluation of the [¹³¹I]2NUBTA using murine sarcoma S180 cells showed increase in radioactivity in hypoxic cells up to 4 h, while it was not in aerobic cells. Its potential as a cerebral ischemia marker was evaluated using gerbil stroke models that had been subjected to right common carotid artery ligation to produce cerebral ischemia. The uptake in the right cerebral hemisphere decreased slowly than that of the left and the right/left hemisphere uptake ratios increased with time going on. It indicated that [¹³¹I]2NUBTA might be a possible cerebral ischemia marker.     

Synthesis and evaluation of novel benzimidazole derivative [Bz-Im] and its radio/biological studies

Two different benzimidazole analogues act as multimodal agent, first one as novel non-peptidic CCK-B receptor antagonist and similarly as potent anti-fungal agent, designated as [Bz-Im]. These compounds were synthesized and characterized by spectroscopic techniques such as FT-IR, NMR, EI-MS and also evaluated for specific radiopharmaceuticals. Preliminary radiolabeling results with (99m)Tc and biological evaluation studies showed promising results for further evaluation in vivo. The efficiency of labeling was more than 97% and complex was stable for about 12h at 30 degrees C in the presence of serum. Both ligands showed binding to most of the organs, known to express CCK receptors in biodistribution studies. Cholecystokinin (CCK(1) andCCK(2)) receptor binding affinities of these analogues are, IC(50), 0.942+/-0.107 for compound C and 0.665+/-0.211 for compound D in rat pancreatic acini. The anti-fungal activity has shown inhibitory activity against Aspergillus flavus and Aspergillus niger. These studies have provided a new template for further development of non-peptidic ligands for diagnostic and therapeutic purposes of diseases related with CCK receptors as well as anti-microbes.     

生物性状发育的遗传修饰

在生物性状发育过程中,除有遗传基础——基因型的控制以外,另一个必要因素,它包括一般的生长发育条件(如温度、营养等)和遗传景背成分。分析修饰基因及其对非等位基因在性状发育上的影响途径和机制,既阐明修饰基因概念,又讲清其对某非等位基因所决定性状的影响的事实例证。把修饰基因作为某非等位基因的细胞内环境条件之一,显示性状发育的复杂性,客观地反映基因与表型(性状)的关系。     

Occurrence of the 15-hydroxy derivative of dihomogammalinolenic acid in human platelets and its biological effect

Various polyunsaturated fatty acids are oxygenated by platelet lipoxygenase at the n - 9 position. The present paper reports that platelets may also oxygenate dihomogammalinolenic acid (20:3(n - 6)) at the n - 6 position, leading to the formation of substantial amounts of 15-OH-8,11,13-20:3 characterized by its ultraviolet spectrum, HPLC and GC-MS analysis. Its formation was inhibited by aspirin and eicosatetraynoic acid, but not by heneicosatetraynoic acid, a specific inhibitor of platelet lipoxygenase. The time-course of its synthesis was very close to that of 12-OH-8,10-17:2 (HHD), the non-cyclic cyclooxygenase side-product, but different from that of 12-OH-8,10,14-20:3, the platelet lipoxygenase end-product of 20:3 (n - 6). Overall, these results indicate that 15-OH-20:3 could be a cyclooxygenase metabolite generated in an aborted process. Like other monohydroxy derivatives of polyenoic fatty acids, 15-OH-20:3 was able to modulate thromboxane-induced platelet aggregation. The derivative exhibited a biphasic effect on the aggregation. It potentiated at concentrations below 2.10(-7) M and inhibited at higher doses. It is concluded that the potentiating activity might explain at least part of the transient enhancement of the platelet activation observed in adding exogenous 20:3(n - 6).     

Solution structures and biological functions of the antimicrobial peptide, arenicin-1, and its linear derivative

Arenicin-1 (AR-1) is a novel antimicrobial peptide that was isolated from coelomocytes of the marine polychaeta lugworm Arenicola marina and shown to contain a single disulfide bond between Cys3 and Cys20, forming an 18-residue ring [Ovchinnikova, T. V. et al., FEBS Lett 2004, 577, 209-214]. To determine the role of this disulfide bond, we synthesized AR-1 (RWCVYAYVRVRGVLVRYRRCW) and its linear derivative, arenicin-1-S (AR-1-S: RWSVYAYVRVRGVLVRYRRSW). Activity assays revealed that AR-1-S is somewhat less active against bacterial cells than AR-1. Both peptides were very hydrophobic, and displayed cytotoxicity against human red blood cells. Analysis of the tertiary structures of AR-1 and AR-1-S by NMR spectroscopy disclosed that AR-1 has two-stranded antiparallel beta-sheet structures with amphipathicity, while AR-1-S displays a random coil structure in DMSO. Our biological data for AR-1 and AR-1-S indicate that the hydrophobic-hydrophilic balance, disulfide bridge, and the amphipathic beta-sheet structure of the peptides play important roles in their biological activities. Elucidation of the structure of AR-1 and its derivative should facilitate the design of novel non-cytotoxic peptide antibiotics with potent antibacterial activities.     

Determination of beta-phenylethylamine as its isothiocyanate derivative in biological samples by gas chromatography mass spectrometry

A detailed procedure of a gas chromatographic mass spectrometric assay for beta-phenylethylamine in biological samples, after its reaction with carbon disulphide to form the isothiocyanate derivative, is presented. Our method has advantages over the previous methods with the pentafluoropropionic derivative of beta-phenylethylamine in that the isothiocyanate derivative of beta-phenylethylamine is much more stable than the pentafluoropropionic derivative and that the background in selected ion monitoring is very low. Using the present method, the levels of beta-phenylethylamine in human urine, untreated and pargyline-treated rat brain were found to be 15.3 micrograms 24 h-1, 1.4 and 160 ng g-1 wet weight, respectively.     

Synthesis and evaluation of a radioiodinated trisaccharide derivative as a synthetic substrate for a sensitive N-acetylglucosaminyltransferase V radioassay

N-acetylglucosaminyltransferase V (GnT-V) is one of the most relevant glycosyltransferases to tumor invasion and metastasis. Based on previous findings of molecular recognition between GnT-V and synthetic substrates, we designed and synthesized a p-iodophenyl-derivatized trisaccharide, 2-(4-iodophenyl)ethyl 6-O-[2-O-(2-acetamido-2-deoxy-β-D-glucopyranosyl)-α-d-mannopyranosyl]-β-D-glucopyranoside (IPGMG, 1) and its radiolabeled form, [(125)I]IPGMG ([(125)I]1), for use in assays of GnT-V activity in vitro. The tributyltin derivative, 2-[4-(n-tributylstannyl)phenyl]ethyl 6-O-[2-O-(3,4,6-tri-O-acetyl-2-acetamido-2-deoxy-β-D-glucopyranosyl)-3,4,6-tri-O-acetyl-α-D-mannopyranosyl]-2,3,4-tri-O-acetyl-β-D-glucopyranoside (21), was synthesized as a precursor for the preparation of [(125)I]1. The iododestannylation of 21 using hydrogen peroxide as an oxidant followed by deacetylation yielded [(125)I]1. When [(125)I]1 was incubated in GnT-V-expressing cells with a UDP-GlcNAc donor, the production of β1-6GlcNAc-bearing IPGMG (IPGGMG, 2) was confirmed by radio-HPLC. In kinetic analysis, 1 was found to be a good substrate with a K(m) of 23.7 μM and a V(max) of 159 pmol/h. μg protein. [(125)I]1 would therefore be a useful synthetic substrate for the quantitative determination of GnT-V activity.     

壳聚糖的血液相容性

壳聚糖是一种天然的氨基多糖,作为一种海洋生物活性物质被广泛地应用于生物医学工 程领域。从血小板、红细胞、白细胞、凝血因子以及补体系统等方面对壳聚糖与血液中各成分的 作用进行了探讨,认为壳聚糖的止血作用是通过激活外源性血液凝固途径和补体系统旁路途径 实现的。在此基础上介绍了几种提高壳聚糖材料血液相容性的方法及相应的抗凝血机理,包括 磺酸化、酰化、表面修饰等。     

Chemical and biological properties of a fluorescence derivative of gentamycin

By reaction with 4-phenylspiro[furan-2(3H),1'-phthalan]-3,3'-dione a fluorescing gentamycin derivative was produced which is characterized by the following properties: High fluorescence intensity in UV light, which enables simple and sensitive determination up to 10(-9) M. In this way it is possible to determine the localization of this substance in tissues, cells and body fluids. When favorably stored, the derivative is chemically stable. At pH 8 and storage at +5 degrees C in the dark for 27 days the substance loses only 5% of its original fluorescence. In comparison with the nonsubstituted gentamyzin the derivative has a twofold higher binding capacity to serum albumin. The antimicrobial properties are retained; the range of antimicrobial effects is similar to that of gentamycin.     

Computer-aided structure analysis of an epimerized dehydroepiandrosterone derivative and its biological effect in a model of reactive gliosis

The naturally occurring steroid dehydroepiandrosterone (DHEA) is reported to reduce glial fibrillary acidic protein (GFAP) overexpression in a model of reactive gliosis due to its conversion to estradiol by the enzyme aromatase. In the present study we examined the biological effect of a new epimerized derivative of DHEA, 16α-iodomethyl-13α-dehydroepiandrosterone derivative (16α-iodomethyl-13α-DHEAd, 16α-iodomethyl-13α-androst-5-en-3β,17β-diol), using the same model system, and compared the 3D structure of this molecule with that of DHEA and two steroidal type aromatase inhibitors, formestane and exemestane. The synthetic compound, in contrast to the reported effect of DHEA, was able to reduce GFAP overexpression only if the enzyme aromatase was inhibited. Data obtained from computational calculations fortified by X-ray crystallography revealed that contrary to the nearly planar sterane framework of DHEA, the synthetic derivative 16α-iodomethyl-13α-DHEAd has a bent sterane skeleton, resulting in a 3D structure that is similar to that of formestane or exemestane. Moreover, 16α-iodomethyl-13α-DHEAd resulted to be metabolically more stable than DHEA.The results suggest that epimerization of the sterane skeleton of DHEA inclines the plane of the D ring, leading to a significantly altered biological activity. The synthetic molecule has a DHEA-like effect on GFAP overexpression when the enzyme aromatase is inhibited and the naturally occurring DHEA is ineffective in this respect. On the other hand, based on their structural similarity it can be hypothesized that 16α-iodomethyl-13α-DHEAd applied alone might have a biological effect similar to that of formestane or exemestane.     

褪黑激素生理特性及应用

对褪黑激素的产生、合成、检测方法、对生殖的调控和应用及最新的研究成果等作了综述。     

A diphenylmethane derivative selective for the anti-estrogen binding site may help define its biological role

By employing as a probe the new compound, N,N-diethyl-2-[(4-phenyl-methyl)-phenoxy]-ethanamine X HC1 (N,N-DPPE), which preferentially binds the anti-estrogen binding site, it is demonstrated that this site appears to contribute to the growth inhibitory action of tamoxifen on MCF-7 human breast cancer cells, even at lower concentrations of this anti-estrogen (1 X 10(-7) M to 1 X 10(-6) M) at which the major effect is clearly mediated via estrogen receptor. The combination of N,N-DPPE and tamoxifen is additive and this effect is not abolished by 17 beta-estradiol. This suggests that the anti-estrogen binding site is not simply a passive reservoir for binding tamoxifen, but may itself mediate the cytotoxic effects of specific ligands.     

Biochemical and biological characterization of a novel anti-aromatase coumarin derivative

Estrogen stimulates the proliferation of estrogen receptor (ER)-positive breast cancer cells. Aromatase is the enzyme responsible for the conversion of androgens into estrogens, and synthetic aromatase inhibitors such as letrozole, anastrozole, and exemestane have proven to be effective endocrine regimens for ER-positive breast cancer. In a recent study, we have found that 4-benzyl-3-(4'-chlorophenyl)-7-methoxycoumarin is a potent competitive inhibitor of aromatase with respect to the androgen substrate. Its K(i) value was determined to be 84 nm, significantly more potent than several known aromatase inhibitors. The specific interaction of this compound with aromatase was further demonstrated by the reduction of its binding by several mutations at the active site region of aromatase and evaluated by computer modeling analysis. The structure-activity studies have revealed that three functional groups (i.e. 3-(4'-chlorophenyl), 4-benzyl, and 7-methoxyl) of this coumarin are important in its inhibition of aromatase. In addition, through a matrigel thread three-dimensional cell culture, this compound was shown to behave like known aromatase inhibitors that suppress the proliferation of aromatase and estrogen receptor positive MCF-7aro breast cancer cells. This coumarin has been shown not to be cytotoxic at up to 40 mum. It was found not to be an inhibitor of steroid 5alpha-reductase that also utilizes androgen as the substrate and not to be a ligand of ERalpha, ERbeta, estrogen-related receptors, or androgen receptor. These results demonstrate that coumarins (a common type of phytochemical) or their derivatives can be potent inhibitors of aromatase and may be useful in suppressing aromataseand ER-positive breast tumors.