Stable transfection of the oestrogen receptor gene into a human osteosarcoma cell line

The oestrogen receptor (ER) gene was introduced into an ER-negative osteoblast-like osteosarcoma cell line HTB 96 by transfection. A number of clones were isolated which expressed ER at levels of up to 70 fmol/mg cytosol protein as determined by immunoassay. Scatchard analysis of the binding of [3H]17 beta-oestradiol in cytosols demonstrated the presence of high affinity binding sites, with a dissociation constant of 0.08-0.13 nM at 4 degrees C. High levels of a 3 kb ER mRNA are produced by the clones, which have gene copy numbers ranging from 2 to greater than 10. Functional receptor activity has been demonstrated by co-transfection of a plasmid containing the chloramphenicol acetyl transferase (CAT) gene linked to an oestrogen response element. Induction of CAT activity is observed in the presence of added oestradiol and is concentration-dependent. The transfected ER is also able to affect endogenous cellular function as several ER-positive clones, but not HTB 96 cells, are growth inhibited by oestradiol in the concentration range 10(-9)-10(-7) M. These effects on growth are not induced by other classes of steroids and are reversible by antioestrogens. No endogenous genes have yet been identified which are oestrogen-regulated in ER-transfected clones.     

Metabolism of halogenated bisphosphonates by the cellular slime mould Dictyostelium discoideum.

Methylenebisphosphonate and its monofluoro-, difluoro- and dichloro- derivatives inhibited growth of amoebae of Dictyostelium discoideum. Dichloromethylenebisphosphonate was the most potent inhibitor of amoebal growth whereas difluoromethylenebisphosphonate was the least potent inhibitor. Each of the bisphosphonates was taken up by the amoebae and incorporated into the corresponding beta, gamma-methylene analogue of adenosine triphosphate. Two of the bisphosphonates were also incorporated into the corresponding analogues of diadenosyl tetraphosphate. No correlation was found between the ability of the bisphosphonates to inhibit amoebal growth and the extent to which they were metabolised.     

Ecology of gorillas and its relation to female transfer in mountain gorillas

Understanding the principles that underly primate social evolution depends on integrated analysis of data on behavioral ecology, demography, life history tactics, and social organization. In this paper, data on the behavioral ecology of gorillas are reviewed and comparisons made among the three subspecies. Gorillas are selective feeders; and, their patterns of food choice are consistent with models of feeding by large generalist herbivores. They rely heavily on terrestrial herbaceous vegetation, which provides an abundant supply of densely distributed food. Availability of this food varies little in space and time; and, gorilla foraging activity can maintain its productivity. The level of frugivory and the extent of seasonal variation in diet and habitat use vary among and within populations. Low variability in food distribution patterns makes cooperative defense of foraging areas not worthwhile; but, it also means that ecological costs associated with gregariousness are low. However, demographic and life history data on mountain gorillas show that these costs may be sufficient to reduce female reproductive success as group size increases. Advantages to being with high quality males apparently can outweigh these costs. The implications of these data for the evolution of the mountain gorilla social system, and the possible roles of male protection, predation, and female/female competition in this regard, are discussed.     

Isolation and characterization of a membrane-attack-complex-inhibiting protein present in human serum and other biological fluids.

Cytosolic, detergent-solubilized and membrane-bound growth hormone (GH) receptors from rabbit adipose tissue and liver were tested for reactivity with a panel of monoclonal antibodies (MAbs). The cytosolic and detergent-solubilized forms of adipose tissue and liver GH receptors were identically reactive with four precipitating and two hormone-binding-site-directed MAbs. However, the membrane-bound form of the adipose receptor was 1000-fold less reactive with one binding-site-directed MAb (MAb 7) than the membrane-bound liver GH receptor. Reactivity with another inhibitory MAb (MAb 263) was identical for adipose tissue and liver membrane GH receptors. The relative potency of 22,000-Mr and 20,000-Mr forms of human GH was identical in assays with liver and adipose tissue membrane receptors. Thus, contrary to earlier suggestions, the discrepancy between the growth-promoting and insulin-like activities of 20,000-Mr human GH cannot be rationalized by a difference in the affinity of this hormone for 'somatogenic' and 'metabolic' receptors when the comparison is made in the same species. Cross-linking studies showed that the major GH-binding subunit of liver and adipose tissue GH receptors had the same Mr (54,000 +/- 5000, reduced). The ligand-binding subunits of liver and adipose tissue receptors are identical by several criteria, but one epitope on the adipose tissue receptor appears to be masked upon membrane insertion, possibly by close association with a tissue-specific component. Tissue specificity may be determined by association of a ubiquitous GH-binding subunit with tissue-specific membrane components, rather than by differences in amino acid sequence.     

Autoradiographic comparison of [I]LSD-labeled 5-HT2A receptor distribution in rat and guinea pig brain

Although the density and distribution of 5-HT_2A(5-hydroxytryptamine-2A) receptors is well established for rat brain, the 5-HT_2A receptor distribution and density in guinea pig brain has not been extensively studied. In the present in vitro study, we have utilized ¹²⁵I-lysergic acid diethylamide ([¹²⁵I]LSD) to quantify and compare 5-HT_2A receptor density in coronal sections of rat and guinea pig brain. Spiperone (1 μM) and sulpiride (1 μM) were used to displace [¹²⁵I]LSD binding from 5-HT_2A and D₂ binding sites, respectively. Ligand binding was quantified by computer-aided image analysis densitometry (MCID). Similar to the rat, areas of highest specific 5-HT_2A receptor binding (fmol/mg protein) in guinea pig brain included the claustrum and Layer 4 of the cerebral cortex. Significant binding was also found in remaining neocortical layers, islands of Calleja, caudate putamen, olfactory bulb, nucleus accumbens, and choroid plexus. While the rat brain exhibited a high level of specific binding in the tenia tecta and mammillary nuclei, little binding was observed in these regions in the guinea pig. In both rat and guinea pig, low specific binding was found in amygdaloid, thalamic, or cerebellar areas. These studies indicate a general similarity between 5-HT_2A binding site distribution and relative density in guinea pig and rat brain but point to a few brain regions where significant differences exist.     

DNA-binding induces a major structural transition in a type I methyltransferase.

The type IC DNA methyltransferase M.EcoR124I is a complex multisubunit enzyme that recognizes the non-palindromic DNA sequence GAAN6RTCG. Small angle X-ray scattering has been used to investigate the solution structure of the methyltransferase and of complexes of the enzyme with unmethylated and hemimethylated 30 bp DNA duplexes containing the specific recognition sequence. A major change in the quaternary structure of the enzyme is observed following DNA binding, based on a decrease in the radius of gyration from 56 to 40 A and a reduction in the maximum dimension of the enzyme from 180 to 112 A. The structural transition observed is independent of the methylation state of the DNA. CD shows that there is no change in the secondary structure of the protein subunits when DNA is bound. In contrast, there is a large increase in the CD signal arising from the DNA, suggesting considerable structural distortion which may allow access to the bases targeted for methylation. We propose that DNA binding induces a large rotation of the two HsdM subunits towards the DNA, mediated by hinge bending domains in the specificity subunit HsdS.     

Molecular response of the lipid headgroup to bilayer hydration monitored by 2H-NMR.

The effect of hydration on the conformation and dynamics of the phosphatidylcholine headgroup has been investigated by 2H-NMR measurements of liquid crystalline dioleoylphosphatidylcholine in multilamellar liposomes. Deuterium quadrupole splittings (delta nu Q) and spin-lattice relaxation rates (1/T1) were recorded for three selectively labeled headgroup segments (alpha, beta, and gamma) over the range of water/lipid mole ratios from 4 to 100. The smooth changes in delta nu Q and 1/T1 are found to essentially parallel each other and can be described by a single exponential decay function. Progressive hydration thus induces a concerted change in headgroup conformation together with an increase in its rate of motion (detected by delta nu Q and 1/T1, respectively). The enhanced mobility is partially due to a shift in the lipid phase transition temperature (as monitored by differential scanning calorimetry) and is furthermore attributed to an entropic contribution. It is concluded that the choline dipole becomes slightly raised in its average orientation into the aqueous layer and that the rate is increased at which the headgroup is fluctuating and protruding. The observed molecular changes can thus be accommodated within a model where the effective accessible headgroup volume expands with increasing hydration.     

Floral expression of a gene encoding an E2-relatedubiquitin-conjugating protein from Arabidopsis thaliana

An Arabidopsis thaliana gene (UBC6) encoding a homologue to ubiquitin-conjugating enzymes has been isolated which is capable of encoding a protein of 183 amino acids of ca. 21 kDa. Northern analysis indicates that the gene is expressed in flowers, seeds and, to a somewhat lesser extent, in 10-day seedlings but not in mature leaves, callus and pre-flowering plants. This pattern of expression is confirmed using transgenic Arabidopsis plants containing a UBC6 promoter-GUS gene fusion construct. These plants displey GUS activity in mature anthers prior to dehiscence, in developing embryos, sepals and the style after pollination.