Development of a simplified, sensitive high-performance liquid chromatographic method using fluorescence detection to determine the concentration of UCN-01 in human plasma

UCN-01 is a naturally derived anticancer agent isolated in the culture broth of actinomyces streptomyces. We have developed a sensitive high-performance liquid chromatographic method for the determination of UCN-01 in human plasma. UCN-01 was isolated from human plasma after intravenous administration, by using 100% ice-cold acetonitrile liquid–liquid phase extraction. Liquid chromatographic separation was achieved by isocratic elution on a phenyl analytical column. The mobile phase consisted of acetonitrile–0.5 M ammonium acetate (45:55) with 0.2% triethylamine added as a modifier. The UCN-01 peak was identified from other peaks using fluorescence excitation energy and emission energy wavelengths of 310 and 410 nm, respectively. Retention time for UCN-01 was 4.2±0.5 min. The UCN-01 peak was baseline resolved, with nearest peak at 2.6 min distance. No interfering peaks were observed at the retention time of UCN-01. Peak area amounts from extracted samples were proportional over the dynamic concentration range used: 0.2 to 30 μg/ml. Mean recoveries of UCN-01 at concentrations of 0.5 and 25 μg/ml were 89 and 90.2%, respectively. Relative standard deviations for UCN-01 calibration standards ranged from 1.89 to 2.31%, with relative errors ranging from 0.3 to 11.6%. Assay precision for UCN-01 based on quality control samples of 0.50 μg/ml was ±4.86% with an accuracy of ±5.7%. For drug extracted from plasma the lowest limit of detection was 0.1 μg/ml, with the lowest limit of quantitation being 0.2 μg/ml. This method is suitable for routine analysis of UCN-01 in human plasma at concentration from 0.2 to 30 μg/ml.     

Requirement for heparan sulphate proteoglycans to mediate basic fibroblast growth factor (FGF-2)-induced stimulation of Leydig cell steroidogenesis

This study reports that, in contrast to previous findings, basic fibroblast growth factor (FGF-2) stimulates immature Leydig cell steroidogenesis in the absence of luteinizing hormone (LH). Heparan sulphate proteoglycans (HSPGs) are essential for this action of FGF-2 and the data suggest that HSPG/FGF-2 interactions have a significant role in the maintenance of immature Leydig cell steroidogenesis. Culture conditions were established for the maintenance of immature rat Leydig cells steroidogenesis in vitro for at least 2 days. Under these conditions the effect of exposure to FGF-2 at doses ranging from 0.1–10 ng/ml was shown to cause a significant stimulation of basal, but not LH-stimulated, 5-androstane 3,17β-diol production over 24h in culture. This stimulatory action on basal steroidogenesis is mediated through HSPG, as it was blocked by the addition of heparin (100 μg/ml), sodium chlorate (25mM) and protamine sulphate (5 μg/ml). These data demonstrate the involvement of HSPG in regulating FGF-2 action on Leydig cells and a potential role for Leydig cell HSPG in mediating paracrine regulatory actions of other heparin binding growth factors.     

Simplified Culture and Chromosome Preparations of Primate Leukocytes

Plasma recovered from 1 ml of primate peripheral blood by centrifugation is planted in a medium consisting of 80% TC-199 and 20% fetal bovine serum, to which 0.125 ml of phytohaemagglutinin/5 ml is added. The pH is adjusted to 7 with 10% NaHCO₂. The mixture is incubated 68 hr, Colcemide to give 1 μg/ml is added, and incubation continued for 4 hr. Following centrifugal separation, the cells are given a hypotonic treatment with 0.75% sodium citrate for 15 min, then centrifuged again and fixed in 3:1 methanol-glacial acetic acid, 3 changes. Tiny drops of the cell suspension are placed on a slide, spread by blowing, and air dried. The preparations are stained with 15% Giemsa solution in methyl alcohol. The method has been successfully used in 256 specimens from 25 different species.     

Effects of cordycepin on macromolecular synthesis and development in the preimplantation mouse embryo

Investigations were conducted to test the effects of cordycepin, a naturally-occurring analog of adenosine, on gene activity in preimplantation mouse embryos. Embryos were explanted into culture at the 2-cell, morula and blastocyst stages, and incubated in the absence or presence of cordycepin (5–100 μg/ml) to determine the effects of the drug on continued development and macromolecular synthesis. Cordycepin at concentrations exceeding 10 μg/ml caused a dose-responsive inhibition of cleavage and blastulation of embryos in culture. Exposure of morulae and blastocysts to cordycepin concentrations of 10–100 μg/ml produced a dose- and time-dependent suppression of RNA synthesis as measured by incorporation of [³H]uridine. Suppression in blastocyst-stage embryos was enhanced by preincubation, and reached 70% after 4 h at 100 μg/ml. Cordycepin (50–100 μg/ml) reduced synthesis of major RNA components detected by electrophoresis, blocked incorporation of radioactivity into fractions bound by olido(dT)-cellulose, and produced a time- and dose-dependent reduction of protein synthesis in blastocysts, causing a maximum inhibition of 25% after 4 h of preincubation at 50 μg/ml.     

Effects of genistein and lavendustin on reproductive processes in domestic animals in vitro

The aim of our experiments was to study the influence of genistein [tyrosine kinase (TK) inhibitor with estrogenic activity] and lavendustin A (TK inhibitor without estrogenic activity) on female reproductive processes in domestic animals in vitro. It was found that genistein (0.001–1 μg/ml) increased IGF-I release by cultured bovine and porcine granulosa cells, but decreased its secretion by rabbit granulosa cells (0.01–10 μg/ml). Genistein stimulated progesterone secretion by bovine and rabbit granulosa cells (at 0.01–10 μg/ml), estradiol output by rabbit granulosa cells (at 1 μg/ml) and porcine ovarian follicles (at 10 μg/ml), as well as cAMP production by bovine (at 0.001–1 μg/ml) and rabbit (at 1 μg/ml) granulosa cells. No effects of genistein (at 10 μg/ml) on PGF-2 alpha and progesterone release by porcine ovarian follicles were observed. Genistein significantly (P < 0.05) stimulated the reinitiation and completion of nuclear maturation of porcine oocytes (at 5 μg/ml), as well as the preimplantation development of rabbit zygotes (at 1 μg/ml). Lavendustin A (0.001–1 μg/ml) increased IGF-I release by bovine (but not by porcine) granulosa cells, cAMP release by bovine granulosa cells, and PGF-2 alpha output by porcine ovarian follicles (at 10 μg/ml). Lavendustin (at 1 μg/ml) had no significant effect on IGF-I release by porcine granulosa cells, on estradiol and cAMP output by rabbit granulosa cells, or on progesterone secretion by porcine follicles (at 10 μg/ml). Inhibitory actions of lavendustin (at 10 μg/ml) on estradiol secretion by porcine follicles were also found. Furthermore, lavendustin, like genistein, promoted the reinitiation and completion of meiosis in porcine oocytes. The present study demonstrates a predominantly stimulatory effect of TK inhibition on endocrine and generative processes in domestic animals. The majority of these effects are similar for both compounds, indirectly suggesting that their action is due to tyrosine kinase inhibition and protein kinase A-stimulation, rather than estrogenic activity.     

Involvement of steroid hormones in bovine oocytes maturation in vitro.

Influences of steroid hormone additions or of their binding by specific antisera on nuclear maturation and subsequent fertilization and cleavage of bovine oocytes were studied in vitro. It was found that progesterone in doses of 50 ng/ml, 250 ng/ml, 1 μg/ml or 5 μg/ml stimulates reinitiation and in doses of 1 or 5 μg/ml stimulates further development of meiosis. Antiserum to progesterone had opposite effects on nuclear maturation, but has no influence on the ability of matured oocytes to subsequent fertilization and cleavage. Testosterone additions (10 ng, 100 ng, 1 μg or 5 μg/ml) did not influence nuclear maturation, but antiserum to this hormone inhibited both meiosis reinitiation and completion, as well as lowered the rate of oocytes fertilized and embryos obtained. Estradiol (5, 50, 100 or 500 ng or 5 μg/ml) treatment stimulated reinitiation, but not nuclear maturation. Antiserum to estradiol activated both reinitiation, development and completion of meiosis, but the cells matured by estradiol deficit were as a rule uncapable of fertilization and further cleavage. Estradiol addition (1 μg/ml) to maturation medium together with FSH (10 μg/ml) (but not of FSH alone) lead to a significantly higher rate of fertilization and cleavage of matured cells.

Results obtained suggest (1) relative independence of reinitiation, further development of nuclear maturation and cytoplasmic maturation regulation in bovine oocytes as well as (2) the involvement of steroid hormones in these three processes.     

An Air-Drying Technique for Flattening Chromosomes in Mammalian Cells Grown In Vitro

The method described was developed to facilitate the analysis of chromosome complements in cells freshly isolated from monkey kidney cortex and grown on glass, and in “altered” monkey cells grown on glass or in suspension. Cells were treated with hypotonic solution (quarter-strength Tyrode or diluted medium) for 30 min, or with colchicine in a final concentration of 25 μg/ml (.0025%) for 12-18 hr followed by hypotonic salt solution for 5 min, then fixed in acetic alcohol (1:3) for 5 min. With cells centrifuged from suspended cultures, addition of fixative had to be gradual. Directly after fixation, films of cells on slides were air dried completely. This produces a more uniform and complete flattening of cells than can be achieved by manual pressure; yet, fragmentation of chromosome complements does not occur. Fixed and air dried slides may be stored for days without deterioration or they may be stained immediately in 2% natural orcein (G. T. Gurr, London) in 50% acetic acid. Preparations can be made permanent by a dry ice schedule, without loss, shrinkage, or distortion of cells.     

Determination of vigabatrin in human plasma and urine by high-performance liquid chromatography with fluorescence detection

A sensitive and specific HPLC method has been developed for the assay of vigabatrin in human plasma and urine. The assay involves derivatization with 4-chloro-7-nitrobenzofurazan, solid-phase extraction on a silica column and isocratic reversed-phase chromatography with fluorescence detection. Aspartam was used as an internal standard. The assay was linear over the concentration range of 0.2–20.0 μg/ml for plasma and 1.0–15.0 μg/ml for urine with a lower limit of detection of 0.1 μg/ml using 0.1 ml of starting volume of the sample. Both the within-day and day-to-day reproducibilities and accuracies were less than 5.46% and 1.6%, respectively. After a single oral dose of 500 mg of vigabatrin, the plasma concentration and the cumulative urinary excretion of the drug were determined.     

Microspike-mediated particle transport towards the cell body during early spreading of 3T3 cells

Freshly trypsinized 3T3 cells send out microspikes of 0.2 μm diameter and up to 10 μm length within 20 min after attachment to a glass substratum. The microspikes move actively and eventually attach to the substratum. Subsequently, lamellae flow out between lines of attached microspikes. If, however, colloidal gold particles of 0.2–0.4 μm diameter and clusters of gold particles up to 4 μm in diameter are placed on the substratum and a microspike attaches to them, we observed two reactions of the microspikes to this contact. They either retract upon contact, transporting the attached particles to the cell surface at a speed of 0.2 μm/sec, or the particles flow toward the cell body while the microspike stays in place. This action results in the clearing of a circular area around each spreading cell before lamellae flow out. “Clearing” proceeds at serum concentrations between 1 and 20% and in concentrations of colchicine up to 20 μm/ml. In concentrations of cytochalasin B higher than 5 μg/ml, however, particle removal is completely inhibited, although the microspikes are still produced by the cell. Transmission electron microscopy shows that the microspikes contain mostly longitudinally oriented microfilaments and only a few microtubules, if any.     

Obtaining Suspensions of Animal Cells in Metaphase from Cultures Propagated on Glass

Cells of some strains continuously cultivated in vitro have a pronounced tendency to lose their attachment to glass during mitosis. If division of these cells is arrested in meta-phase by treatment with colchicine, many of the cells are set free and float into the medium. Advantage has been taken of this property to collect large numbers of suspended cells in c-metaphase. By applying standard procedures of staining with orcein and manual squashing to these cells, preparations are obtained that are useful for critical chromosome analysis. The procedure consists in treatment of cultures with colchicine (final concentration 0.1 μg/ml) for 18-24 hr; agitation in hypotonic medium (quarter-strength Tyrode's or CMRL-1066) for 5 min; addition of 1:3 acetic-alcohol for 5-10 min; removal and centrifugation of the resulting fluid mixture from the culture; and resuspension of the sedimented cells in a small volume of the fluid. Advantages of the method are: large numbers of metaphase plates can be examined on a single slide, the chromosomes are contracted and well separated, and cytoplasmic boundaries are preserved.     

Liquid chromatographic determination of sodium mercaptoundecahydrododecaborate in rat urine and plasma after precolumn derivatization

A high-performance liquid chromatographic (HPLC) method was developed for the determination of disodium mercaptoundecahydrododecaborate (BSH) in biological fluids. Monobromobimane was used as a precolumn derivatizing agent. A stable derivative was obtained. The derivative was separated on a C₁₈ column using reversed-phase ion-pairing chromatography and detected by a spectrophotometric detector at 373 nm. The detection limit was 200 ng/ml (0.1 ppm boron). Calibration curves were prepared for rat urine and plasma samples. The calibration curves were linear in the range of 1 μg/ml to 100 μg/ml for urine samples and 0.2 μg/ml to 50 μg/ml for plasma samples.     

Chromosome Preparations in the Field from Mammals Long After Death

Chromosome preparations of high quality can be obtained from bone marrow cells of small mammals that have been dead for 20 hr or longer. The bone marrow is rinsed out of the femurs with RPMI medium supplemented with 15% fetal calf serum. Add 0.05-0.1 ml of a 0.01 % colchicine solution to 5 ml of medium-cell suspension. After Vi-1 hr of colchicine treatment at 37 C the cells are spun down and the supernatant replaced by 5 ml of hypotonic (0.075 M) KC1. After 12 min in the hypotonic solution at 37 C the cells are fixed in methanokacetic acid 3:1. Air dried preparations are made after repeating the fixation procedure three times and the chromosomes are stained with Gietnsa, if required after prclieatment of the preparations for banding; e.g., GTG. Technical hints for field work are given. The technique has proven successful with several species of rodents and shrews.     

Comparison between aseric and seric culture-derived exoantigens of Babesia divergens in their ability to induce immunoprotection in gerbils

Babesia divergens Rouen 1987 was cultivated with a high percentage of parasitized erythrocytes (30–40%) in either RPMI 1640 supplemented by 10% human serum or in a serum-free medium consisting of RPMI 1640 supplemented with 5 g/l Albumax I®. Analysis of serum and Albumax culture supernatants, using polyacrylamide gel electrophoresis, revealed the presence of at least 10 parasitic exoantigens of B. divergens with molecular weights ranging between 27 and 200 kDa. The gerbils were injected twice, at 3-week intervals, with Albumax culture supernatants or seric culture supernatants. The vaccine doses ranged from 3 μl to 1.5 ml. The highest immunofluorescent antibody titers in gerbils (in 42 days) were obtained using Albumax supernatant and Quil A saponin as adjuvant. Analysis of the gerbil humoral response by immunoprecipitation showed that only three exoantigens were immunodominant: 92 kDa, 50 kDa and 37 kDa proteins. The gerbils were challenged 3 weeks after the last vaccine injection and the maximum protection was observed with vaccine doses ranging from 30 μl to 1.5 ml of culture supernatant and Quil A adjuvant. Albumax medium-derived antigens potentiated better protection at lower dose rates than that of serous medium-derived antigens (for example the gerbil mortality was 0% when they are immunized with 30 μl of Albumax supernatant and 100% with 30 μl of seric supernatant).     

柞蚕核型多角体病毒载体在培养细胞和休眠蛹中的基因表达效果

柞蚕核型多角体病毒（AnpeNPV）作为基因表达载体在柞蚕培养细胞（AnPe细胞）和柞蚕蛹中已经成功地表达出了外来基因,并生产出了大量蛋白质。本文比较了AnpeNPV与苜蓿尺蠖核型多角体病毒(AcMNPV)、家蚕核型多角体病毒(BmNPV)和美国白蛾核型多角体病毒(HycuNPV)基因表达载体在培养细胞和昆虫活体组织内的β-半乳糖苷酶基因表达效果。结果显示,5×105个细胞中β-半乳糖苷酶的最高酶活性分别是AnpeNPV在AnPe细胞为40.9 units/ml （TC-100培养液,FBS10%）和59.9 units/ml（SF-900Ⅱ培养液）,AcMNPV在Sf9细胞为72.4 units/ml（TC-100,FBS10%）和66.4 units/ml（SF-900Ⅱ）、在High5细胞为326 units/ml（EX-CELL 405培养液）,BmNPV在Bm4细胞为15.1 units/ml（TC-100,FBS10%）,HycuNPV在SpIm细胞为68.6 units/ml（SF-900Ⅱ）。活体组织内β-半乳糖苷酶的最高酶活性分别是柞蚕雌蛹为14.3 units/g、雄蛹为11.7 units/g,家蚕幼虫是10.1 units/g。实验证明AnpeNPV/AnPe的外来基因表达水平与AcMNPV/ Sf9和HycuNPV/SpIm相似、比BmNPV/ Bm4高、不及AcMNPV/ High5;AnpeNPV/柞蚕蛹,其雌蛹比BmNPV/家蚕5龄幼虫的外来基因表达效果好、雄蛹与之无明显差异,说明AnpeNPV基因表达载体无论是在培养细胞还是昆虫活体组织中均可与其他NPV基因表达载体相媲美。柞蚕蛹由于可以机械化、大规模地操作,显示对于大量生产蛋白质具有更好的应用前景。     

A Method for Chromosome Preparation of Sea Urchin Embryos

Chromosome preparations of the sea urchin Clypeaster japonicus were made from early embryos. To obtain well spread chromosomes in air dried preparations, fertilization membranes were removed, embryos treated with colcemid (0.1-2.0 μg/ml) were dissociated into their component cells by Ca-Mg-free sea water, and dissociated cells were swollen with hypotonic (0.5 M) KCl. Thanks to adequate spreading of the cytoplasm, only the chromosomes stained well with Giemsa.     

Chromosome preparations in the field from mammals long after death

Chromosome preparations of high quality can be obtained from bone marrow cells of small mammals that have been dead for 20 hr or longer. The bone marrow is rinsed out of the femurs with RPMI medium supplemented with 15% fetal calf serum. Add 0.05-0.1 ml of a 0.01% colchicine solution to 5 ml of medium-cell suspension. After 1/2-1 hr of colchicine treatment at 37 C the cells are spun down and the supernatant replaced by 5 ml of hypotonic (0.075 M) KCl. After 12 min in the hypotonic solution at 37 C the cells are fixed in methanol:acetic acid 3:1. Air dried preparations are made after repeating the fixation procedure three times and the chromosomes are stained with Giemsa, if required after pretreatment of the preparations for banding, e.g., GTG. Technical hints for field work are given. The technique has proven successful with several species of rodents and shrews.     

Chromosome Preparatons of Bone Marrow Cells without Prior In Vitro Culture or In Vivo Colchicine Administration

Bone marrow (about 0.5 ml) from au erythropoietic region is freed of blood clots by washing 1-3 min in 1 μg/ml colchicine solution (2-3 ml) and then soaking 1-2 hr at 20-30° C in a second change. For mammalian or avian marrows, the colchicine is made up in phosphate-buffered (pH 7) physiological NaCl solution; for amphibian, Ringer's A solution. Next the specimens are soaked about 20 min in a hypotonic solution as follows: for mammalian, 1% Na-citrate; for avian, a 1:4 dilution of the buffered NaCl solution by distilled water; and for amphibian, Ringer's A-distilled water, 1:1. Then they are heated in a mixture of 2% orcein in 45% acetic acid and 1 N HCl, 9:1. Immediately after heating, squash preparations are made with 2% acetic-orcein in the usual manner. An alternative method is to dissociate the marrow cells by agitating after colchicine treatment. Then, recovering the cells between changes by low-speed centrifugation, to carry out the hypotonic treatment and subsequent fixation in Carnoy's solution I (alcohol acetic, 3:1) before drying the cells onto slides from the fixative. After thorough drying the slides may be stained 10-20 min in acetic orcein, or by other suitable technics.     

High-density lipoproteins decrease both binding of a polynuclear aromatic hydrocarbon carcinogen to DNA and carcinogen-initiated cell transformation

Lipophilic carcinogens partition into the three major classes of lipoproteins potentially present in serum used as a medium supplement for cell culture. Different serum lots sequester differing quantities of the procarcinogen benzo[a]pyrene, dependent on the serum lipoprotein concentrations. In the presence of high-density lipoproteins a mutagenic benzo[a]pyrene metabolite was bound to cellular DNA at decreased levels when compared to cells exposed to the mutagen in the absence of high-density lipoproteins. Fetal calf serum with low levels of lipoproteins, specifically, high-density lipoproteins, is associated with efficient methylcholanthrene-initiated transformation of C3H/10T1/2 cells, while calf serum with a significant concentration of high-density lipoproteins requires up to a 500% increase in methylcholanthrene concentration to achieve similar levels of transformation in this mouse embryo cell line. When concentrated serum lipoproteins or purified HDL were added to fetal calf serum containing MCA at μg/ ml, the C3H/10T1/2 transformation frequency was decreased compared to the transformation frequency achieved in the presence of 1 μg/ml of MCA in fetal calf serum without supplementation. The results suggest that high-density lipoprotein partitioning of lipophilic polynuclear aromatic hydrocarbon mutagens from the cell culture medium may effectively reduce the concentration of carcinogen available for interaction with cellular DNA in vitro, which, in turn, may be associated with decreased carcinogen-induced transformation of cells.     

In vitro antiprotozoal activity of the lipophilic extracts of different parts of Turkish Pistacia vera L.

Thirteen lipophilic extracts prepared with n-hexane from various parts of Pistacia vera L. tree (Anacardiaceae) growing in Turkey were screened for their in vitro activity against four parasitic protozoa, Trypanosoma brucei rhodesiense, Trypanosoma cruzi, Leishmania donovani and Plasmodium falciparum. Melarsoprol, benznidazole, miltefosine, artemisinin and chloroquine were used as reference drugs. The cytotoxic potentials of the extracts on rat skeletal myoblast (L6) cells were also assessed and compared to that of podophyllotoxin. The screening method employed was medium-throughput, where the extracts were tested at two concentrations, at 0.8 and 4.8 μg/ml (T. brucei rhodesiense, L. donovani and Plasmodium falciparum), or at 1.6 and 9.7 μg/ml (T. cruzi and L6 cells). At 4.8 μg/ml concentration, the branch extract of Pistacia vera (PV-BR) significantly inhibited (77.3%) the growth of L. donovani, whereas the dry leaf extract (PV-DL) was active against Plasmodium falciparum (60.6% inhibition). The IC₅₀ values of these extracts were determined as 2.3 μg/ml (PV-BR, L. donovani) and 3.65 μg/ml (PV-DL, Plasmodium falciparum). None of the extracts possessed cytotoxicity on mammalian cells.