A test using cultured cells with induced mixed-function oxygenase in the unscheduled DNA synthesis assay for detecting promutagens/procarcinogens

The human FL cell line contains very low levels of constitutive AHH activity, but it could be greatly induced by NE, beta-NF and 3-MC, and induced slightly by PB. When two different types of inducer, for example, 3-MC and PB or 3-MC and NE were given in combination, an additive inductive effect was not observed. Both the constitutive and induced AHH in FL cells have characteristics of MFO, namely, NADPH-dependence and CO-sensitivity. The fact that the constitutive and induced AHH in FL cells could be inhibited by a known hydroxylase inhibitor 7,8-BF indicated that the AHH in FL cells belongs to the cytochrome P-448 dependent MFO type. After removal of inducer from the medium, the induced AHH activity remained at a high level for at least 24-36 h. By using AHH-induced FL cells in the UDS assay system for the detection of promutagens/procarcinogens, we found that AFB1 and 3-MC did not induce a UDS reaction in uninduced FL cells, while in beta-NF induced cells, 10(-6)-10(-4) M AFB1 and 10(-7)-10(-6) M 3-MC elicited a very significant UDS reaction, which was concordant with the results obtained in the UDS assay system using HeLa cells or FL cells supplemented with liver microsomes or using primary cultured hepatocytes as indicator cells. B(a)P elicited the UDS reaction at concentrations of 10(-6)-10(-3) M in beta-NF induced cells, whereas 10(-4)-10(-3) M was required in uninduced cells. The results above indicate that this new design is feasible, but further study is needed to assure its accuracy.     

Increased thorn length in Acacia depranolobium —an induced response to browsing

Summary I report here longer thorns induced by large mammal herbivory on the tree Acacia depranolobium. I compared trees that had been browsed by domestic goats to trees protected from goat browsing. Thorns on browsed branches within the reach of goats (<125 cm above the ground) were significantly longer than thorns from higher branches on the same browsed trees, and significantly longer than branches at similar heights on unbrowsed trees. It appears that increased thorn length was an induced response to large mammal herbivory in Acacia depranolobium, both among and within individual trees.     

Characterization of capsid and noncapsid proteins of B19 parvovirus propagated in human erythroid bone marrow cell cultures.

The major capsid and noncapsid proteins of the pathogenic parvovirus B19, propagated in vitro, were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoprecipitation, and immunoblot of the erythroid fraction of infected human bone marrow cell cultures. There were two capsid proteins of 58 kilodaltons (kDa; the major species) and 84 kDa (the minor species). Newly synthesized capsid viral proteins were present in the supernatants of infected cultures. The major noncapsid protein of 77 kDa was localized to the nucleus.     

Polyphosphate Formation from Pyrophosphate in Intact Cells of a Photosynthetic Bacterium, Rhodospirillum rubrum

The hypothesis that the photosynthetic bacterium, Rhodospirillumrubrum, may synthesize polyphosphate using pyrophosphate asa precursor has been tested. The cells were cultured in thepyrophosphate media, and the cell yield, cell shape, and thepolyphosphate content were determined and compared with thatgrown in the orthophosphate media. In the control experiment, the total amount of polyphosphateformed by the cultures grown in the pyrophosphate media wasfound to be up to 70% less than that grown in orthophosphatemedia. When the ATP formation was blocked by using the phosphorylationinhibitor, antimycin A, however, the cells grown in the pyrophosphatemedia formed up to 50% more polyphosphate than that grown inthe orthophosphate media. The results seem to support the hypothesis that Rhodospirillumrubrum may use pyrophosphate as the precursor of polyphosphate. (Received May 26, 1986; Accepted February 21, 1987)     

Control by insulin and insulin-related growth factor 1 of protein synthesis in a cell-free translational system from chick-embryo fibroblasts.

Insulin and insulin-related growth factor 1 (IGF-1) increase by 1.5-1.6-fold the rate of [3H]leucine incorporation into protein in primary monolayer cultures of chick-embryo fibroblasts (CEF); half-maximal hormone concentrations are 10 and 0.25 nM respectively. To investigate the mechanism of this effect, a rapid method is used to prepare a lysate from CEF which is active in protein synthesis. Lysate derived from cells treated for 30-150 min with insulin synthesized protein at 1.8-3.0-fold greater rate than did controls; the increased rate persisted for 20 min in vitro. Pactamycin (0.5 microM), an inhibitor of peptide-chain initiation, inhibited protein synthesis by 50% in lysates derived from insulin-treated and control cells. Thus insulin and IGF-1 cause an increase in the protein-synthesis rate in vivo, which persists in cell-free protein-synthesizing lysates of CEF.     

Vasomotor control: functional hyperemia and beyond

Historically, functional hyperemia has been viewed largely as an interaction between a parenchymal cell and its associated microvasculature. Locally released metabolites have been thought to produce relaxation of the smooth muscle and a vasodilation that increases blood flow in proportion to metabolic need. This symposium report presents evidence from a variety of disciplines and a number of different types of biological preparations that demonstrates that functional hyperemia is a complex process involving several classes of microvessels including capillaries, arterioles, and small arteries. These vessels do not function independently but are coordinated by a complex set of interrelations involving at least three different modes of interaction between parenchymal cells and the various segments of the vascular bed. These are local metabolic effects, propagated effects extending over long segments of the vasculature, and flow-dependent vasodilation induced by local changes in blood flow. In addition to these acute responses to metabolic demand it appears that tissues may be capable of more long-term structural alterations of the arterial and arteriolar network in response to sustained changes in the relationship between supply and demand. The vascular bed appears to be able to adapt either by increasing the maximal anatomic diameter of the large arteries or by inserting new arterioles into the parenchyma. Thus, classical functional hyperemia appears to be but one manifestation of a multifaceted process leading to highly coordinated responses of many vascular elements, resulting finally in vascular patterns that are optimized to meet parenchymal cell demands.     

Nucleotide sequence of a gene cluster involved in entry of E colicins and single-stranded DNA of infecting filamentous bacteriophages into Escherichia coli.

Mutations in fii or tolA of the fii-tolA-tolB gene cluster at 17 min on the Escherichia coli map render cells tolerant to high concentrations of the E colicins and do not allow the DNA of infecting single-stranded filamentous bacteriophages to enter the bacterial cytoplasm. The nucleotide sequence of a 1,854-base-pair DNA fragment carrying the fii region was determined. This sequence predicts three open reading frames sequentially coding for proteins of 134, 230, and 142 amino acids, followed by the potential start of the tolA gene. Oligonucleotide mutagenesis of each open reading frame and maxicell analysis demonstrated that all open reading frames are expressed in vivo. Sequence analysis of mutant fii genes identified the 230-amino acid protein as the fii gene product. Chromosomal insertion mutations were constructed in each of the two remaining open reading frames. The phenotype resulting from an insertion of the chloramphenicol gene into the gene coding for the 142-amino acid protein is identical to that of mutations in fii and tolA. This gene is located between fii and tolA, and we propose the designation of tolQRA for this cluster in which tolQ is the former fii gene and tolR is the new open reading frame. The protein products of this gene cluster play an important role in the transport of large molecules such as the E colicins and filamentous phage DNA into the bacterium.     

Post-translational processing of the porcine gastrin precursor by phosphorylation of the COOH-terminal fragment

The gene sequence encoding porcine preprogastrin is known; in order to clarify pathways of post-translational processing of the predicted precursor peptide we have characterized material reacting with antibodies to a synthetic peptide corresponding to the expected extreme COOH-terminal portion of the precursor. Radioimmunoassay was used to identify and monitor the purification of peptides in porcine antral mucosa. Two peptides (I and II) were isolated to homogeneity by steps involving gel filtration, ion exchange, and reversed-phase high performance liquid chromatography. The two co-eluted on gel filtration but were separated on anion-exchange chromatography. The more acidic peptide (II) was less hydrophobic on high performance liquid chromatography. Automated gas-phase microsequencing revealed the less acidic peptide (I) to have the sequence of porcine preprogastrin 96-104 (SAEEGDQRP); it would be produced by tryptic-like cleavage of Arg95-Ser96. The second peptide did not yield a phenylthiohydantoin-derivative on the first cycle but thereafter it sequenced as the first peptide (i.e. -AEEGDQRP). Incubation in alkali liberated almost equimolar amounts of phosphate from peptide II but not from I. In addition, alkaline phosphatase liberated phosphate and converted the acidic peptide to the less acidic one. The results suggest that serine in the first position is phosphorylated in peptide II but not I. The tripeptide -Ser(P)-Ala-Glu- also occurs in adrenocorticotropic hormone; this tripeptide is a substrate for physiological casein kinase. Potential phosphorylation sites occur at comparable positions in the precursors of a number of regulatory peptides.