Solubilisation and reconstitution of acylcoenzyme A:estradiol-17 beta acyltransferase

Acylcoenzyme A:estradiol-17 beta acyltransferase in microsomes of bovine placenta cotyledons was strongly membrane bound. The enzyme was solubilised from microsomes by sodium cholate and was reconstituted into phospholipid vesicles. The apparent Km for estradiol-17 beta was 11 microM which was close to the value of 8 microM previously found with the membrane-bound enzyme. Testosterone was also a substrate for the reconstituted enzyme (apparent Km 62 microM) and was a competitive inhibitor (Ki 74 microM) of the acylation of estradiol-17 beta. Although various long-chained fatty acyl CoAs acted as acyl donors, these proved to have widely differing apparent Km values with palmitoleoyl CoA having the highest affinity (Km 24 microM) and arachidonoyl CoA the lowest affinity (Km 330 microM).     

An antibody to the receptor for insulin-like growth factor I inhibits the growth of MCF-7 cells in tissue culture

Alpha IR-3, a monoclonal antibody to the insulin-like growth factor I receptor which blocks insulin-like growth factor I binding and inhibits its activity, inhibits the binding of 125I-insulin-like growth factor I to MCF-7 cells (an estrogen dependent human breast carcinoma cell line) with an IC-50 of approximately 100 ng/ml. It also inhibits the growth of MCF-7 cells cultured in 5% calf serum with approximately the same IC-50. Inhibition of growth occurs both when cells are cultured in the presence and absence of estrogen and is more pronounced when cells are grown at a low density. These findings demonstrate a requirement for insulin-like growth factor I for optimal growth of MCF-7 cells and suggest that it is an autocrine growth factor in these cells.     

The production of hybrid Ty:IFN virus-like particles in yeast.

The yeast retrotransposon Ty encodes proteins that assemble into virus-like particles (Ty-VLPs) which can be readily purified. We have recently shown that expression of the pl protein encoded by the TYA gene of Ty is sufficient for particle formation. In this paper we show that when a heterologous coding sequence, human interferon-alpha 2 (IFN), is fused in frame to the TYA gene, the resulting p1-IFN fusion protein is still assembled into VLPs. These Ty:IFN-VLPs can be easily purified to near homogeneity and furthermore, they induce an antibody response to interferon when they are injected into rabbits. Therefore, these data show that hybrid Ty-VLPs can be used as a convenient system for the efficient purification of fusion proteins in yeast.     

Seasonal variation in the diet of the king penguin (Aptenodytes patagonicus) at sub-Antarctic Marion Island

King penguins are important consumers of marine resources, throughout the year, at the Prince Edward Islands. Meal size varied from 8.5–12.6% of adult mass, depending on the method of determination. In spite of the biases in the analysis favouring the overestimation of squid, fish and, in particular, myctophid fish accounted for the largest proportion of the stomach samples, 87% by wet mass, 75% by numbers and 69% by reconstituted mass. The relative abundance of fish in the diet dropped markedly in winter followed by a subsequent rise to nearly 100% in summer. This rise coincided with an increase in the chick growth rate and the king penguin population at the island and suggests the rise in relative abundance offish reflects a real increase in the availability of fish around the islands.
Juvenile and adult Krefftichthys anderssoni/Protomyctophum tenisoni and adult Electrona carlsbergi were the most common fish consumed. There was an increase in the modal size of K. anderssoni/P. tenisoni throughout the year which we interpret as growth of a single fish population. Juvenile Kondakoviu longimana was the important squid species taken by king penguins. Crustaceans were only rarely recorded in the diet and may have come from digestion of fish and squid stomachs.
This is the first study of the diet of a Southern Ocean pelagic predator that has identified myctophid fish as a major component of its diet. All three important fish species taken by king penguins at Marion Island have a wide distribution throughout the Southern Ocean and consequently may prove to be important dietary components of other Southern Ocean pelagic predators.     

Interactions of three sequentially expressed genes control temporal and spatial specificity in Aspergillus development

Aspergillus nidulans brlA, abaA, and wetA form a dependent pathway that regulates asexual reproductive development. The order in which these genes are expressed determines the outcome of development. Expression of brlA in vegetative cells leads to activation of abaA and wetA, cessation of vegetative growth, cellular vacuolization, and spore formation. By contrast, expression of abaA in vegetative cells does not result in conidial differentiation but does lead to activation of brlA and wetA, cessation of vegetative growth, and accentuated cellular vacuolization. brlA, abaA, and wetA act individually and together to regulate their own expression and that of numerous other sporulation-specific genes. We propose that the central pathway controlling development is largely autoregulatory. The timing and extent of expression of the regulatory genes and their targets are determined as development proceeds by intrinsically controlled changes in the relative concentrations of regulatory gene products in the various conidiophore cell types.     

Oncogene cooperation in lymphocyte transformation: malignant conversion of E mu-myc transgenic pre-B cells in vitro is enhanced by v-H-ras or v-raf but not v-abl.

Although transgenic mice bearing a c-myc gene controlled by the immunoglobulin heavy-chain enhancer (E mu) eventually develop B-lymphoid tumors, B-lineage cells from preneoplastic bone marrow express the transgene but do not grow autonomously or produce tumors in mice. To determine whether other oncogenes can cooperate with myc to transform B-lineage cells, we compared the in vitro growth and tumorigenicity of normal and E mu-myc bone marrow cells infected with retroviruses bearing the v-H-ras, v-raf, or v-abl oncogene. The v-H-ras and v-raf viruses both generated a rapid polyclonal expansion of E mu-myc pre-B bone marrow cells in liquid culture and 10- to 100-fold more pre-B lymphoid colonies than normal in soft agar. The infected transgenic cells were autonomous, cloned efficiently in agar, and grew as tumors in nude mice. While many pre-B cells from normal marrow could also be induced to proliferate by the v-raf virus, these cells required a stromal feeder layer, did not clone in agar, and were not malignant. Most normal cells stimulated to grow by v-H-ras also cloned poorly in agar, and only rare cells were tumorigenic. With the v-abl virus, no more cells were transformed from E mu-myc than normal marrow and the proportion of tumorigenic pre-B clones was not elevated. These results suggest that both v-H-ras and v-raf, but apparently not v-abl, collaborate with constitutive myc expression to promote autonomous proliferation and tumorigenicity of pre-B lymphoid cells.     

Mouse DNA methylase. Intracellular location and degradation

DNA methylase extracted with low salt from mouse Krebs II ascites cell nuclei has been degraded stepwise by trypsin treatment. Degradation, accompanied by a limited reduction in size of the native enzyme, leads to the progressive introduction of several nicks so that, eventually, fragments of 14, 18, 24 and 28 kD are released on denaturation. This illustrates the domain structure of the enzyme. In contrast to ascites cell nuclear extracts, preparations from liver nuclei are already nicked and the major from of the enzyme contains a 100 kD fragment though the native molecular weight is unchanged. Newborn mouse liver contains more undegraded enzyme that is mostly firmly-bound within the nucleus. Trypsin treatment increases the de novo activity of the enzyme and prevents its aggregation in the absence of salt, even in the presence of high concentrations of native DNA.     

Superoxide dismutase inhibition of alkaline hemin-mediated O2 activation

Inhibition of hemin-mediated O2 activation by bovine superoxide dismutase and the copper tetrammine complex has been examined. It is shown that the inhibitory effect of these species is not due to metabolism of O2-., but to a nonspecific inhibition of the process by the Cu2+ ion. We propose that the inhibition occurs at the level of electron transfer from NADH to iron in an analogous manner to that proposed for the Cu2+ inhibition of microsomal electron transfer.