Amplification and rearrangement in hepatoma cell DNA associated with integrated hepatitis B virus DNA.

DNA of hepatitis B virus is found to be integrated into the genome of infected human liver cells and may be related to the development of primary liver carcinoma. We have previously reported the cloning of cellular DNA with integrated HBV sequences from the PLC/PRF/5 cell line which derives from a human primary liver carcinoma. Two clones, designated as A-10.7 and A-10.5, and a third uncloned fragment are compared by restriction enzyme mapping, hybridization and nucleotide sequencing. The results indicate that amplification of integrated viral DNA and host flanking regions has occurred, followed by transposition and/or major deletions. The implications of these findings for the development of primary liver carcinoma are discussed.     

C-Photosynthate Partitioning and Translocation in Soybeans during Reproductive Development

Partitioning and translocation of ¹⁴C-photosynthates were examined during flowering and seed maturation in soybean (Glycine max [L.]Merr.) plants to quantify allocation to sugars, amino acids, organic acids, and starch and to study transport of C and N from leaves to reproductive sinks. The trifoliolate leaf at the eighth node was exposed to steady state levels of ¹⁴CO₂ for 2 hours, followed by immediate extraction and identification of radioactive assimilates in the fed leaf blade, tissues of the transport path (e.g. petiole and stem), and fruits if they were present. About one-third of the total ¹⁴C recovered from the leaf blades was in starch until late pod-filling, after which the proportion dropped to 16%. Sugars comprised 70% to 86% of the recovered ¹⁴C from soluble assimilates of the source leaf, with highest proportions occurring during late flowering and early pod-filling. Amino acids accounted for 8% to 17% of the ¹⁴C recovered from the soluble fraction, and were most evident during early flowering and mid to late pod-filling. The ¹⁴C-organic acids comprised from 3% to 14% of the soluble ¹⁴C-assimilates in leaves. Petioles consistently contained a higher percentage of recovered radioactivity in sugars (87-97%) and a lower percentage in amino acids (3-12%) than did leaf blades. ¹⁴C-Amino acids in petioles attained their highest levels during mid and late pod-filling, while ¹⁴C-organic acids comprised 2% or less of the recovered radioactivity after pod initiation. The distribution of ¹⁴C-assimilates in the internode below the source leaf was similar to that found in petioles. A comparison of the above data to calculated C and N requirements for seed development suggests that ¹⁴C-amino acids derived from current photosynthesis and translocated from source leaves supply at least 12% to 48% of the seed N depending on the stage of pod-filling.     

Localized Photosynthate Deposition in Citrus Fruit Segments Relative to Source-Leaf Position

Photosynthate translocation into fruit segments was examinedin ‘Pineapple’ sweet orange Citrus sinensis (L.)Osbeck to determine whether previously reported patterns ofdistribution [Koch (1984) HortScience 19: 260] would changeover time or with alterations in balance between source leavesand sink fruit. In control plants, ¹⁴CO₂ was supplied to a sourceleaf nearest the fruit for 1 h, followed by 5 h translocation.Over 89% of [¹⁴C]assimilates in the fruit were localized in4 segments directly aligned with the source and 73% of thesewere in the center 2 segments. Peel, pulp and seeds showed similarpatterns. Little or no lateral spreading of [¹⁴C]photosynthatesoccurred when an additional 7 days were allowed for translocation,but distribution was slightly broader when the source leaf was8 nodes farther from the fruit. Defoliation and girdling toreduce the source/sink ratio gave variable results if done 18h before experiments, but widened the area receiving [¹⁴C]assimilatesto approximately half the fruit if done 7 days earlier. Thisoccurred only when an entire fruit, was dependent upon a singlesource, leaving the opposite half fruit without an externalsupply of photosynthates. These data show an extreme degreeof preferential translocation and inflexibility which can occurin a transport path. ¹Supported by United States Department of Agriculture CompetitiveResearch Grant 59-2121-1-1-752-0, Regional Project NC-142, andthe Institute of Food and Agricultural Sciences, Universityof Florida. (Received January 12, 1984; Accepted May 14, 1984)     

Non-aceticlastic methanogenesis from acetate: acetate oxidation by a thermophilic syntrophic coculture

Methanogenesis from acetate by a rod-shaped enrichment culture grown at 60° C was found to require the presence of two organisms rather than a single aceticlastic methanogen. A thermophilic Methanobacterium which grew on H₂/CO₂ or formate was isolated from the enrichment. Lawns of this methanogen were used to co-isolate an acetate oxidizer in roll tubes containing acetate agar. The rod-shaped acetate oxidizer was morphologically distinct from the methanogen and did not show F₄₂₀ autofluorescence. The coculture completely degraded 40 mol/ml acetate, and produced nearly equal quantities of methane, and methanogenesis was coupled with growth. The doubling time for the coculture at 60°C was 30–40 h and the yield was 2.7±0.3 g dry wt/mol CH₄. Studies with ¹⁴C-labelled substrates showed that the methyl group and the carboxyl group of acetate were both converted primarily to CO₂ by the coculture and that CO₂ was concurrently reduced to CH₄. During growth, there was significant isotopic exchange between CO₂ and acetate, especially with thecarboxyl position of acetate. These results support a mechanism for methanogenesis from acetate by the coculture in which acetate was oxidized to CO₂ and H₂ by one organism, while H₂ was subsequently used by a second organism to reduce CO₂ to CH₄. Since the H₂ partial pressure must be maintained below 10^-4 atm by the methanogen for acetate oxidation to be thermodynamically feasible, this is an example of obligate interspecies hydrogen transfer. This mechanism was originally proposed for a single organism by Barker in 1936.     

Molecular species of epidermal growth factor carrying immunosuppressive activity

The suppression of antibody formation to sheep red cells in mice by partially purified fractions of mouse submaxillary gland was shown to be caused by epidermal growth factor (EGF). Purification of EGF by the method of Savage and Cohen resolved three components referred to as EGF a, EGF b, and EGF c. All three induced premature eye opening in neonatal mice, but only EGF a (identified as EGF 1-53) had full immunosuppressive activity. EGF c was shown by micropeptide mapping of chymotryptic and thermolytic digests and amino-terminal analysis to differ from EGF a only by the presence of beta-aspartyl instead of an asparaginyl residue. EGF b differed from EGF a in that it lacked the N-terminal asparagine. EGF shortened enzymatically at its carboxy terminal by two or five amino acids did not have any immunosuppressive activity. These findings suggest that, in contrast to some other biological effects of EGF, intact amino and carboxy terminals are required for the expression of immunosuppressive activity.     

On the role of tryptophan in luteinizing-hormone-releasing hormone (luliberin).

The tryptophan residue of luteinizing-hormone-releasing hormone (luliberin) was chemically modified to produce the following analogs: [Trp(o)3]luliberin, Trp-(2,4-dinitrophenylsulfenyl)-luliberin, Trp-(2-hydroxy-5-nitrobenzyl)luliberin, (Trp-S-luliberin)2, Trp-CH3S-luliberin and Trp-formyl-luliberin. The luteinizing-hormone-releasing activity of those analogs was determined by bioassay in vitro and found to be 0.2%, 0.2%, 0.6%, 1.5%, 1.7% and 7% of that of the natural hormone, respectively. These results demonstrate that alterations in the indole moiety of tryptophan-3, which lead to a reduction in its electron density or sterically restrict its electron avaliability, are associated with a dramatic loss of biological activity.     

Divergent effects of cyanate on amino acid and phosphate uptake by liver and hepatoma.

The uptake of alpha-aminoiso[3H]butyric acid and 32Pi was observed to be inhibited by sodium cyanate in transplanted hepatomas but was increased in the livers of the tumor bearing rats. Incorporation of 32Pi into macromolecules in hepatomas was also inhibited by cyanate. Treatment with this drug did not influence circulating concentrations of isotope-labeled materials. There were relatively small effects on uptake of 36Cl- in cyanate-treated rats and the action was not tissue specific. The data were compatible with an inhibitory effect of cyanate on active transport in hepatomas which was not seen under the same conditions in host liver.     

Aryl sulfatase inactivation of slow reacting substance. Evidence for proteolysis as a major mechanism when ordinary commercial preparations of the enzyme are used

Type II B arylsulfatases are known to inactivate slow reacting substance (SRS), but the mechanism is unclear. In the present study, ordinary commercial preparations of Sigma limpet arylsulfatase largely inactivated the glutathionyl and cysteinyl-glycyl forms of SRS, but the cysteinyl form of SRS was largely resistant to the enzyme. Evidence is presented which established that a major mechanism for the inactivation of the glutathionyl and cysteinyl-glycyl SRS types, at least by the particular enzyme preparations we have studied, involves cleavage of the glycine moiety from the sulfur containing side chain. This was confirmed by digestion studies with glutathione itself. In addition, there is some evidence to indicate that the enzyme may destabilize the double bond structure of the SRS molecule, contributing to the overall inactivation.     

Effect of screening on the incidence of cervical cancer in Alberta.

The rates of registration of cases of in-situ and invasive cancer of the cervix in Alberta have fallen for women aged 35 and over since the introduction of screening in the early 1960s, as predicted by theory and described in Finland. However, for women aged 15 to 34 years of age the predicted pattern was followed only initially: the registration rate for in-situ and probably also invasive cancer increased after 1973. This could be due to an actual increase in the incidence of in-situ cancer of the cervix among younger women, as might be expected from the epidemiologic aspects of the disease, but it might also be due to increased recruitment of younger women to the screening program.