Effect of hypothermia on cell kinetics and response to hyperthermia and X rays

Hyperthermia is a potent radio enhancer. Studies using hypothermia in combination with irradiation have given confusing results due to lack of uniformity in experimental design. This report shows that hypothermia might have potential significance in the treatment of malignant cells with both thermo- and radiotherapy. Reuber H35 hepatoma cells, clone KRC-7 were used to study the effect of hypothermia on cell kinetics and subsequent response to hyperthermia and/or X rays. Cells were incubated at 8.5 degrees C or between 25 and 37 degrees C for 24 hr prior to hyperthermia or irradiation. Hypothermia caused sensitization to both hyperthermia and X rays. Maximum sensitization was observed between 25 and 30 degrees C and no sensitization was found at 8.5 degrees C. At 25 degrees C maximum sensitization was achieved in approximately 24 hr, cell proliferation was almost completely blocked, and cells gradually accumulated in the G2 phase of the cell cycle. In contrast to the effect of hypothermia on either hyperthermia or X rays alone, thermal radiosensitization was decreased in hypothermically pretreated cells (24 hr at 25 degrees C) compared to control cells (37 degrees C). The expression of thermotolerance and the rate of development at 37 degrees C after an initial heating at 42.5 degrees C were not influenced after preincubation at 25 degrees C for 24 hr. The expression of thermotolerance for heat or heat plus X rays during incubation at 41 degrees C occurred in a significantly smaller number of cells after 24 hr preincubation at 25 degrees C. The enhanced thermo- and radiosensitivity in hypothermically treated cells disappeared in approximately 6 hr after return to 37 degrees C.     

Expression of a Rhizobium phaseoli Sym plasmid in R. trifolii and Agrobacterium tumefaciens: incompatibility with a R. trifolii Sym plasmid

Identification of the Sym plasmid in Rhizobium phaseoli strain RCC3622 is described. Introduction of this plasmid into R. trifolii or Agrobacterium tumefaciens strains resulted in bacteria capable of forming characteristic spherical root nodules on beans. This Sym plasmid, designated pSym9, was characterized as 275 MDa and nonconjugative. pSym9 was incompatible with the R. trifolii Sym plasmid pSym5, and carries genes determining a melanin-like black pigment. A second plasmid of 135 MDa, pRph3622a, was also transferred from R. phaseoli to R. trifolii and A. tumefaciens. Transconjugants carrying this plasmid did not form root nodules on beans. In contrast to other Rhizobium plasmids, pRph3622a was unstable in A. tumefaciens.     

The effect of pH and lysosomal extract on rat liver metallothionein (Cd-thionein)

Was studied in vitro. The¹⁰⁹Cd-labeled protein was isolated by gel filtration and incubated with a lysosomal extract from the same source. No degradation was seen when the pH of the incubation medium was 5 or higher, whereas the degradation of Cd-thionein was completed in 2 h at a pH of 4.5. Dissociation of Cd²⁺ from thionein, which takes place to a fair extent at a pH below 5, appears necessary before the enzymatic breakdown of the thionein moiety can take place.     

Fluorescence decay of pyrene in small and large unilamellar L,α-Dipalmitoylphosphatidylcholine vesicles above and below the phase transition temperature

The fluorescence decays of pyrene in small and large unilamellar L,-dipalmitoylphosphatidylcholine vesicles have been investigated as a function of probe concentration and temperature. When the molar ratio of pyrene to phospholipid equals 1:3000, no excimer emission is observed and the fluorescence decays are mono-exponential. When this ratio is equal to or higher than 1:120, excimer formation is observed.Above the phase transition temperature the observed fluorescence decays of monomer and excimer can be adequately described by a bi-exponential function. The monomer decays can be equally well fitted to a decay law which takes into account a time-dependence in the probe diffusion rate constant. The fluorescence decay kinetics are compatible with the excimer formation scheme which is valid in an isotropic medium. The excimer lifetime and the (apparent) rate constant of excimer formation have been determined as a function of probe concentration at different temperatures above the phase transition temperature. The activation energy of excimer formation is found to be 29.4±1.3 kJ/mol. In small unilamellar vesicles the diffusion constant associated with the pyrene excimer formation process varies from 8.0x10^-7 cm²/s at 40°C to 2.2x10^-6 cm²/s at 70°C.Below the phase transition temperature the monomer decays can be described by a decay law which takes into account a time dependence of the rate constant of excimer formation. The lateral diffusion coefficient of pyrene calculated from the decay fitting parameters of the monomer region varies from 4.0x10^-9 cm²/s at 20°C to 7.9x10^-8 cm²/s at 35°C. No significant difference could be observed between the pyrene fluorescence decay kinetics in small and large unilamellar vesicles.Abbreviations SUV small unilamellar vesicles - LUV large unilamellar vesicles - DPPC dipalmitoylphosphatidylcholine - DMPC dimyristoylphosphatidylcholine - FRAP fluorescence recovery after photobleaching Part of this research has been presented at the 5th international symposium on surfactants in solution. Bordeaux, July 9th–13th 1984     

A chimaeric hygromycin resistance gene as a selectable marker in plant cells

Summary We show here that plant cells are sensitive to the antibiotic hygromycin-B⁴. We also show that a chimaeric gene consisting of the nopaline synthase (nos) gene regulatory elements and the E. coli derived hygromycin phosphotransferase (hpt) gene, when transferred to plants' cells, confers resistance to hygromycin B. The chimaeric nos-hpt gene enables efficient selection of DNA transfer to plant cells when used in conjunction with Ti plasmid-derived binary vectors in cocultivation experiments.     

Flow cytometric evaluation of cell dispersion from human head and neck tumors

The preparation of single-cell suspensions from 25 human head and neck tumors is described. Dispersal was performed overnight at 4 degrees C under slight agitation of the tissue suspensions using various combinations of enzymes and additives. The cell suspensions were examined for number of cells released, viability, amount of debris, and DNA distribution by means of flow cytometry (FCM). It was shown that both trypsin/dithioerythritol (TD) and collagenase/D Nase (CDse) were of value in dispersing single cells from tumor tissue. In contrast to CDse, incubation with TD appeared to be cytolytic to normal lymphocytes. In a number of cases, DNA-FCM revealed ploidy abnormalities in a TD-suspension, which were not discernible in the concurrent CDse-suspension. Cell culture of primary cell suspensions corroborated the reliability of the DNA-FCM measurements. Pretreatment with CDse improved tumor disaggregation by TD and indicated a different dispersal capacity. Addition of Ca2+ and Mg2+ ions to the dispersal mixtures and preincubation of tumor slices in complete medium for 1 day before initiation of cell dispersion influenced favorably the quality of the cell suspension.     

Functional aspects of the postovulatory follicle in the ovary of the African catfish,Clarias gariepinus,after induced ovulation

Summary In captive African catfish, Clarias gariepinus, ovulation was induced with human chorionic gonadotropin (HCG) 4 I.U./g body weight to study the function of postovulatory follicles (POFs). Ultrastructural and enzyme-histochemical data indicate that, apart from special theca cells, the granulosa of relative young POFs (i.e., from 16 h and 28 h after HCG-injection) is capable of producing steroids. Possible functions of the synthesized steroids are discussed. Histological comparison of POFs from stripped and from unstripped fish, as well as histochemical investigation of the contents of ovulated ova and granulosa of POFs at 48 h after HCG-injection, showed that the latter structure is involved in phagocytosis of the disintegrating ovulated eggs. The polysaccharide-lipid-protein material, initially taken up by heterophagolysosomes of the granulosa cells, subsequently undergoes fatty degeneration. The granulosa cells of the POFs showed strong acid phosphatase activity and abundant granular endoplasmic reticulum from 16 h after HCG-injection onward; heterophagolysosomes appeared at 32 h. These results indicate that after ovulation the phagocytotic function of the granulosa develops progressively. Autophagolysosomes, responsible for the final disintegration of POFs, become increasingly evident in the granulosa cells with increasing time after ovulation.     

In vitro culture of intramolluscan stages of the avian schistosome Trichobilharzia ocellata

Several different procedures for in vitro cultivation of intramolluscan stages of the avian schistosome Trichobilharzia ocellata were tried. A medium was found and culture conditions were established that not only supported in vitro transformation of miracidia into mother sporocysts, but also resulted in substantial subsequent growth; moreover, some degree of germinative development appeared to occur as well. Cerebral ganglia from uninfected adult snails of the intermediate host species, Lymnaea stagnalis, could produce factors promoting in vitro development of young mother sporocysts. Results are compared with data from the literature and it is concluded that greater success in in vitro culturing of young mother sporocysts of T. ocellata can be achieved than has hitherto been reported for other schistosome species. The same culture procedures were less successful when applied to other intramolluscan stages of T. ocellata, but can be used for in vitro maintenance of these stages. The procedures described here will be a useful tool in the study of schistosome-snail interactions in T. ocellata-L. stagnalis and possibly in other systems as well.     

Phosphatidylglycerol as biosynthetic precursor for the poly(glycerol phosphate) backbone of bifidobacterial lipoteichoic acid.

Phosphatidylglycerol functions as donor of the sn-glycerol 1-phosphate units in the synthesis in vitro of the 1,2-phosphodiester-linked glycerol phosphate backbone of the lipoteichoic acids of Bifidobacterium bifidum subsp. pennsylvanicum. The incorporation was catalysed by a membrane-bound enzyme system. After addition of chloroform/methanol the product formed coprecipitated with protein. The material was phenol-extractable and was co-eluted with purified lipoteichoic acid on Sepharose 6B. The reaction was stimulated by Triton X-100, UDP-glucose and UDP-galactose, but Mg2+ ions had no effect. The apparent values for Km and Vmax. of the phosphatidylglycerol incorporation were 1.4 mM and 3.1 nmol/h per mg of membrane protein, respectively. Labelled UDP-glucose and UDP-galactose were not incorporated into the lipoteichoic acid fraction by the particulate membrane preparation.     

Human c-fms proto-oncogene: comparative analysis with an abnormal allele.

The organization of the human c-fms proto-oncogene has been determined and compared with an abnormal allele. The human v-fms homologous genetic sequences are dispersed discontinuously and colinearly with the viral oncogene over a DNA region of ca. 32 kilobase pairs. The abnormal c-fms locus contains a small deletion in its 3' portion. DNA sequencing analysis indicated that it was 426 base pairs in size and located in close proximity to a putative c-fms exon.