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PubMed1980

Extraction of lipids from mammalian liver using nontoxic solvents.

A simple method for extracting and purifying lipids from rat liver in a single step using nontoxic solvents is described. The method consists homogenizing the pulverized tissue with a mixture of trichlorotrifluoroethane (Cl2CF-CCIF2) and isopropyl alcohol (1 : 1, v/v). Just enough water is added to the lipid extract to produce a biphasic system. Pure lipid extract is obtained by isolating the lower layer from the aqueous upper phase which contains the non-lipid materials. The described method compares favourably with that of Folch et al., both quantitatively. The solvent system used also has the advantage of being less toxic than the widely used chloroform/methanol system, which makes it safer for prolonged use. The new method is simple, efficient and reproducible.

1-Propanol↗
PubMed1984

A method for isolation of undegraded free and membrane-bound ribosomes from rat lactating mammary gland.

Mammary gland polysomes are difficult to isolate from the lactating rat using methods developed for other species and tissues, most likely due to high calcium-stimulated ribonuclease activity in that tissue. A new method, utilizing ethyleneglycol-bis-(beta-aminoethylether)-N,N'-tetraacetic acid (EGTA) to bind calcium, yields highly aggregated polysomes from lactating rat mammary gland. Fresh mammary tissue is pulverized under liquid nitrogen. Free and membrane-bound polysomes are isolated by differential centrifugation in solutions containing 100 mM KCl, 100 mM MgCl2, 75 mM EGTA, 500 micrograms/ml heparin and 50 mM Tris buffer, pH 8.2 at 5 degrees C. Bound polysomes are released from the endoplasmic reticulum using Triton X-100 and deoxycholate. Polysome profiles are obtained on linear sucrose gradients and scanned at 254 nm. The method gives quantitative recovery of homogenate total RNA. To demonstrate that the method can be used to study nutritional effects on mammary gland polysome aggregation, lactating rats were fasted 22-66 h and then refed a stock diet for 71-95 h. Refeeding increased the percentage of polysomes (trimers or larger) in the bound fraction from 84 +/- 1 to 93 +/- 1% (P less than 0.001) and in the free fraction from 42 +/- 2 to 55 +/- 3% (P less than 0.001).

Animals↗
PubMed1982

Double minutes in mouse neuroblastoma cells and their hybrids.

Cytogenetic studies were carried out on three clones of mouse neuroblastoma cells and six interspecific hybrid cells derived from the mouse neuroblastoma cells with either rat glioma cells and liver cells or Chinese hamster brain cells. The hybrid cells possessed characteristic karyotypes with marker chromosomes originating from the neuroblastoma cells. The parental chromosome constitution in the hybrid cells was clone-specific, even in the clones derived from the same parental cells. Double minutes (DMs) were demonstrated in the neuroblastoma cells and in all the hybrid cells studied. In addition other chromosome aberrations, such as microchromosomes and chromosome pulverization, were also observed in these cells. DMs varied in number and morphology among the cells. The number of DMs per cell correlated positively with the level of ploidy and with the karyological constitution contributed by the parental neuroblastoma cells. The results indicate that DMs have a chromosome nature and that the DMs of neuroblastoma chromosomes were transferred into the hybrid cells.

Animals↗
PubMed1989

Increased chromosome breakage by N-methyl-N1-nitro-N-nitrosoguanidine in patients with adenomatous polyposis coli.

In a search for hypersensitivity to chromosome breaking agents, lymphocytes and fibroblasts from patients with the precancerous condition adenomatous polyposis coli (APC) were treated with N-methyl-N1-nitro-N-nitrosoguanidine (MNNG) and bleomycin. The APC cells showed a significantly increased level of chromatid-type damage following MNNG treatment (5 micrograms/ml for lymphocytes, 1 or 2 micrograms/ml for fibroblasts). No such differential effect was noted for bleomycin, but a threefold increase in "pulverized" cells was seen in APC lymphocyte cultures treated in G0, that is before cells have entered the cell cycle. Increased spontaneous and induced chromosome instability appears to be an important effect of the APC mutant gene as molecular evidence suggests that chromosomal mechanisms are likely to play a major role in tumorigenesis both in this condition and in sporadic colorectal cancer.

Adenomatous Polyposis Coli↗
PubMed1990

Acrocentric prophasing in bromodeoxyuridine-incorporated chromosomes.

Chromosomes that appear to be incompletely condensed [pulverized, prematurely condensed chromosomes (PCCs), prophasing] are known to occur in metaphase spreads of cells from normal individuals and more frequently in cells from individuals with malignant disease or in cells exposed in vitro or in vivo to various agents such as viruses, chemicals, and radiation. In this study involving bromodeoxyuridine (BrdU)-treated lymphocytes, a selective prophasing of acrocentrics appeared to be occurring. The acrocentrics involved were generally in interconnected groups.

Bromodeoxyuridine↗
PubMed1991

Cytogenetic analysis of head and neck carcinomas.

Ten primary squamous cell carcinomas (SCC) of the head and neck were evaluated cytogenetically after 10-14 days of in vitro culture. Addition of 3% L-glutamine was essential for consistent epithelial growth of these carcinomas. Outgrowth of cells from tissue explants contained a mixture of chromosomally normal and abnormal cells; the abnormal cells had extensive changes including translocations, marker chromosomes, inversions, deletions, and duplications. In addition, all carcinomas contained cells with pulverization and double minute chromosomes (dmin). Chromosomes 11, 13, and 14 had "hotspots" of rearrangements.

Adult↗
PubMed1993

Chromosome 9 anomalies as the primary clonal alteration in a case of squamous cell carcinoma of the epiglottis.

We present the cytogenetic characterization of a short-term culture of a primary squamous cell carcinoma of the epiglottis from a 67-year-old male patient who was admitted to this hospital for treatment. This patient had a history of chronic exposure to tobacco and alcohol, environmental carcinogens known to be related to the etiology of the disease. The tumor karyotype showed three distinct clones: 1) cells with chromosome 9 anomalies; 2) cells with chromosome 9 and other clonal structural anomalies involving chromosomes 1, 11, 14, and 17; and 3) cells whose chromosomes were partially or totally pulverized. The anomalies on chromosome 9 were homozygous inversion (p12q13), deletions at regions q22, q34.1, and p13, and i(9q) formation. Structural clonal anomalies on other chromosomes included translocations, deletions, and isochromosome formation. The presence of a chromosome 9 anomaly alone or in conjunction with other anomalies suggests that this aberration may be a nonrandom primary event in the progression of squamous cell carcinoma of the head and neck.

Aged↗
PubMed1993

Cytogenetic analysis of human hepatocarcinoma cell line PLC-PRF-5 and its mutant clones with different degrees of cell differentiation.

A detailed analysis was made of the karyotype of human hepatocarcinoma cell line PLC-PRF-5 containing an integrated hepatitis B viral genome and 10 mutant clones derived from the line. These clones are drug resistant and display features of cell differentiation. Cytogenetic manifestations of gene amplification common to many cells resistant to drugs were not observed in these clones, but certain tendencies of karyotype evolution involving material from chromosomes 7 and 11, as well as chromosomes 5 and 15, were recorded for a group of cytostatic-resistant clones. An increase in polyploid cell number and number of cells with chromosome pulverization as compared with the original line was noted in the clones. This phenomenon may be related to the cytopathic effect of the hepatitis B viral proteins, which have a higher expression in the clones.

Carcinoma, Hepatocellular↗
PubMed1982

Chromosome-breaking ability of arecoline, a major betel-nut alkaloid, in mouse bone-marrow cells in vivo.

The chromosomal damaging properties of arecoline, a major betel-nut alkaloid were investigated in vivo after its administration to mice. Arecoline was injected in doses of 0.25, 0.5, 1 and 2 mg for 10, 20 and 30 days each. Significant numbers of aberrations were observed in the form of chromatid breaks, chromosome breaks, ring structure, multiple breaks and cells with pulverized chromosomal complements. The frequency of aberrations showed a dose-response relationship. So arecoline was concluded to have weak chromosome-damaging effects in vivo.

Animals↗
PubMed1985

Cytogenetic analysis of peripheral blood lymphocytes in glass workers occupationally exposed to mineral oils.

Chromosome aberration tests were carried out in a group of 31 pressed glass makers operating an automatic line of press-and-blow machines known to release mineral oil mists containing relatively high concentrations of the mutagenic chemicals belonging to a class of polycyclic aromatic hydrocarbons (PAH). The workers were exposed to the mineral oil aerosol levels that did not exceed the Czechoslovak maximum allowable concentration limit of 5 mg . m-1 of air. The tests revealed that the frequency of aberrant cells (% AB.C.) and the value of breaks per cell (B/C) ratio found in mineral oil-exposed workers were increased significantly, accounting for 4.65 +/- 0.29% AB.C. (0.0532 B/C) vs. 1.13 +/- 0.19% AB.C. (0.0113 B/C) seen in matching controls. Also, a higher rate of dicentrics, reciprocal translocations and cells with pulverization was observed in this group of glass workers. These finding are considered as evidence suggesting that these workers might experience an increased risk of genetic injury due to exposure to mineral oil mists.

Adult↗
PubMed1986

Herpes simplex virus gene products involved in the induction of chromosomal aberrations.

The effect of short-term herpes simplex virus type 1 (HSV-1) infection on chromosomes of human diploid fibroblasts was examined. In addition to chromosomal breaks, gaps and pulverization, three kinds of cytogenetic damage (double minutes, polyploidy and endoreduplication) not yet reported following productive infection with HSV or other animal viruses were frequently observed. Consistent with previous studies suggesting that the expression of immediate-early and/or early viral gene products is required for the induction of chromosomal damage, was the observation that cells infected at the nonpermissive temperature with HSV-1 temperature-sensitive mutants defective in the gene for the immediate-early transcriptional regulatory protein, ICP4, and three early viral gene products--DNA polymerase (pol), the major HSV DNA-binding protein (ICP8) and an HSV-2 mutant defective in alkaline nuclease--exhibited altered patterns of chromosomal damage relative to the effects of wild-type virus on infected cells. These findings suggest a direct or indirect role for all four gene products in the induction of chromosomal damage. In cells infected with wild-type virus for 4 h or longer, HSV proved to be a more potent mitotic arresting agent than colcemid. Moreover, studies with selected mutants indicate that HSV pol specifically may be involved in mitotic arrest. Additionally, in cells infected at the non-permissive temperature with a pol mutant, the number of polyploid metaphases was reduced 4-fold relative to that seen in wild-type virus-infected cells suggesting a role for HSV pol in the amplification of cellular DNA.

Cell Line↗
PubMed1991

Phage typing and phage induction in carrier and invasive Staphylococcus epidermidis isolates.

Fifty-nine Staphylococcus epidermidis isolates (30 carrier and 29 invasive) were phage typed using the phage set developed by Pulverer. Poor results were obtained as only 24% of the invasive and 27% of the carrier isolates were typable, with no differences between these percentages (P greater than 0.05). Isolation of lysogenic phages present in the isolates was attempted by three different methods, induction being satisfactory only by overnight incubation with 0.1 mg l-1 mitomycin C and detection with 2,3,5-triphenyl-tetrazolium chloride. All isolates produced phages, and the lytic activity of these phages was expressed on more than 50% of the isolates in all cases (except one phage which lysed 10% of the carrier isolates and 13.8% of the invasive isolates). The percentages of isolates lysed in the two groups (carrier and invasive) were statistically different (P less than 0.05) only for phages derived from four isolates.

Bacteriophage Typing↗
PubMed1990

The accumulation of metals and their toxicity in the marine intertidal invertebrates cerastoderma edule, Macoma balthica, Arenicola marina exposed to pulverised fuel ash in mesocosms.

In order to investigate the accumulation of metals and related biological effects from Pulverized Fuel Ash (PFA), three intertidal benthic invertebrates were used in exposure studies with different mixtures of PFA sediments. After the first run of 90 days, high mortality was found in the lugworm Arenicola marina. After intermittent exposure to PFA, high mortality was also found for the cockle Cerastoderma edule. No mortality occurred with the baltic tellin Macoma balthica. Metal accumulation differed widely among the species. A. marina accumulated As to high levels in PFA mixtures, which may be attributed to changes in the redox potential of the sediment. It also appeared from this study that Zn levels in M. balthica tissue, for both control and exposed animals, are apparently normal but extremely high. Disposal of PFA in marine coastal waters will radically affect community structure at the dumping site. Accumulation of certain elements like As will be favoured in the mixing zone at the borders of a dump site due to a higher organic content and consequent higher bioavailability.

Journal Article↗
PubMed1987

HPLC micromethod for simultaneous measurement of estradiol, progesterone, androgen and glucocorticoid receptor levels. Application to breast cancer biopsies.

Estradiol (ER), progesterone (PR), androgen (AR) and glucocorticoid receptor (GR) levels were assayed in 25 breast cancer tumors. The tissue was pulverized and homogenized in buffer, then divided into two parts: one was assayed by the standard dextran-coated charcoal method (DCC), with Scatchard plot analysis, the other was assayed by a micromethod developed in our laboratory, as described below: --incubation of the cytosol with several ligands (labelled and unlabelled) selected to avoid unwanted cross-reactions --DCC separation, followed by extraction of all receptor-bound steroids by precipitation of proteins with methanol/TCA --separation of these steroids on a high pressure liquid chromatography (HPLC) column using a methanol/water solvent --collection of the fractions of the column outlet and counting. Use of three labelled ligands and appropriate unlabelled ligands allowed assays of the four receptors. This micromethod was highly correlated with the standard method: ER = 0.985 (P less than 0.001); PR = 0.999 (P greater than 0.001); AR = 0.989 (P less than 0.001); GR = 0.867 (P less than 0.001). Thresholds of positivity were not modified. This micromethod allowed simultaneous measurement of several receptors in 40 mg biopsy specimens and can be applied to other hormone-dependent tissues.

Breast Neoplasms↗
PubMed1984

Comparative toxicities of dietary caffeine and theobromine in the rat.

Caffeine, incorporated into pulverized Purina Rat Chow at a concentration of 0.5%, was fed to male Sprague-Dawley rats for 7 or 8 wk and the effects were compared with those of 0.8% dietary theobromine, fed to male rats for 7 wk. Both dietary methylated xanthines produced significant decreases in food consumption and body-weight gain when compared to their respective control groups. Food consumption of caffeine-fed rats was 57.2% of controls and for theobromine-fed rats it was 77.9% of the respective controls. Theobromine produced significant decreases in thymus weights, with caffeine producing smaller decreases. The theobromine-fed rats showed severe testicular atrophy with extensive spermatogenic cell degeneration and necrosis, while the testes of rats fed caffeine for 7 or 8 wk showed only scattered vacuolar degeneration of spermatogenic cells. Caffeine appears to be more potent than theobromine as an anorexic agent in rats, but to be equivalent to theobromine in its potential for inducing thymic atrophy and spermatogenic cell destruction with testicular atrophy.

Animals↗
PubMed1992

Determination of vinca alkaloids in mouse tissues by high-performance liquid chromatography.

A high-performance liquid chromatographic method is described for the determination of vinblastine in various normal mouse tissues, such as lung, heart, liver, kidney and muscles, and in implanted MO4 tumours. Vincristine was used as the internal standard. Freshly obtained mouse tissue or tumour tissue was frozen at -20 degrees C and then lyophilized. After lyophilisation, the dry tissues were pulverized and homogeneously mixed, and an aliquot was suspended in 0.1 M hydrochloric acid. The drugs of interest were then isolated from this suspension using ion-pair extraction at pH 3 with octylsulphate as counter-ion. The obtained extracts were analysed on a reversed-phase system with a cyanopropyl stationary phase. The detection limit was 1 ng/l in plasma and 10 ng/g in tissue. The extraction recoveries of vincristine and vinblastine were between 45 and 67%, and there were no interferences from blank components. Preliminary pharmacokinetic data for different mouse tissues and tumour implanted in muscle tissue are presented.

Animals↗
PubMed1995

The antiamoebic effect of a crude drug formulation of herbal extracts against Entamoeba histolytica in vitro and in vivo.

The antiamoebic effect of a crude drug formulation against Entamoeba histolytica was studied. In the traditional system of medicine in India, the formulation has been prescribed for intestinal disorders. It comprises of five medicinal herbs, namely, Boerhavia diffusa, Berberis aristata, Tinospora cordifolia, Terminalia chebula and Zingiber officinale. The dried and pulverized plants were extracted in ethanol together and individually. In vitro amoebicidal activity was studied to determine the minimal inhibitory concentration (MIC) values of all the constituent extracts as well as the whole formulation. The formulation had a MIC of 1000 micrograms/ml as compared with 10 micrograms/ml for metronidazole. In experimental caecal amoebiasis in rats the formulation had a curative rate of 89% with the average degree of infection (ADI) reduced to 0.4 in a group dosed with 500 mg/kg per day as compared with ADI of 3.8 for the sham-treated control group of rats. Metronidazole had a cure rate of 89% (ADI = 0.4) at a dose of 100 mg/kg per day and cured the infection completely (ADI = 0) when the dosage was doubled to 200 mg/kg per day. There were varying degrees of inhibition of the following enzyme activities of crude extracts of axenically cultured amoebae: DNase, RNase, aldolase, alkaline phosphatase, acid phosphatase, alpha-amylase and protease.

Acid Phosphatase↗
PubMed1993

Simultaneous determination of drugs of abuse (opiates, cocaine and amphetamine) in human hair by GC/MS and its application to a methadone treatment program.

A new method was developed for the simultaneous detection and quantitation of 6-acetyl-morphine (MAM), amphetamine, benzoylecgonine (BZE), cocaine, codeine, dihydrocodeine, EDDP (methadone metabolite), methadone and morphine in hair. The hair samples were washed, cut into 2-cm segments, pulverized, incubated with phosphate buffer and beta-glucuronidase/aryl-sulfatase. After solid phase extraction and derivatization with pentafluoropropionic anhydride/pentafluoropropanol, the drugs were identified and measured by gas chromatography/mass spectrometry using their deuterated analogues as internal standards. The method is reproducible with detection limits under 0.1 ng/mg hair for almost all substances tested. Fifteen hair samples from five subjects of a methadone treatment program were collected in a 6-month period. The hair samples were segmented and examined for methadone, its main metabolite EDDP, and drugs of abuse. Of the 96 segments analysed, 95% were positive for methadone (mean value, 10.9 ng/mg), 76% for the metabolite EDDP (mean value, 1.2 ng/mg), 69% for opiates (mean values, MAM, 7.3 ng/mg; morphine, 2.9 ng/mg; codeine, 1.0 ng/mg) and 43% for cocaine (mean values, cocaine, 2.6 ng/mg; BZE, 1.1 ng/mg). A correlation of 0.63 was found between administered methadone dosages and concentrations measured by hair analysis. Further investigation is needed to clarify interindividual differences.

Adult↗