The effects of interleukins 1α and 1β on prostaglandin production by cultured human fetal membranes

The effects of IL-1α and IL-1β on cultured human fetal membranes were studied. These cytokines are known to regulate prostaglandin synthesis by the separated components of the fetal membranes (amnion, chorion and decidua), but their effects on intact tissue are unknown. IL-1α increased PGE2 levels on the fetal side of the membrane, indicating increased production of prostaglandin from the amnion, but had little effect on levels of PGE2 on the maternal side of the membrane. Low levels of IL-1β (0.1 - 1.0 ng/ml) increased PGE2 levels on the fetal side of the meembrane, and also increased the production of PGE2 metabolites and PGF2α, suggesting that this cytokine stimulated the decidua as well as the amnion. High concentrations of both cytokines appeared able to stimulate prostaglandin production by the side of the membrane opposing that to which they were added, but it is not clear whether this was mediated by factors released by the stimulated membrane, or by direct transfer of small quantities of cytokines through the membrane. Taken together, these results indicate that IL-1β was a potent stimulator of the synthesis of prostaglandins by decidua and by amnion, whereas IL-1α only stimulated the amnion.     

Structural engineering of the HIV-1 protease molecule with a beta-turn mimic of fixed geometry.

An important goal in the de novo design of enzymes is the control of molecular geometry. To this end, an analog of the protease from human immunodeficiency virus 1 (HIV-1 protease) was prepared by total chemical synthesis, containing a constrained, nonpeptidic type II' beta-turn mimic of predetermined three-dimensional structure. The mimic beta-turn replaced residues Gly16,17 in each subunit of the homodimeric molecule. These residues constitute the central amino acids of two symmetry-related type I' beta-turns in the native, unliganded enzyme. The beta-turn mimic-containing enzyme analog was fully active, possessed the same substrate specificity as the Gly16,17-containing enzyme, and showed enhanced resistance to thermal inactivation. These results indicate that the precise geometry of the beta-turn at residues 15-18 in each subunit is not critical for activity, and that replacement of the native sequence with a rigid beta-turn mimic can lead to enhanced protein stability. Finally, the successful incorporation of a fixed element of secondary structure illustrates the potential of a "molecular kit set" approach to protein design and synthesis.     

Mouse chromosome 1

Chair of Committee for Mouse Chromosome 1     

THE NORMAL FINE STRUCTURE OF OPOSSUM TESTICULAR INTERSTITIAL CELLS

The interstitial tissue of the opossum testis includes interstitial or Leydig cells, macrophages, and small cells which morphologically resemble mesenchymal cells. The latter are thought to give rise to mature interstitial cells. The most prominent feature of the interstitial cell cytoplasm is an exceedingly abundant agranular endoplasmic reticulum. This reticulum is generally in the form of a meshwork of interconnected tubules about 300 to 450 A in diameter, but occasionally it assumes the form of flattened, fenestrated cisternae resembling those of pancreatic acinar cells, except for the lack of ribonucleoprotein particles on the surface of the membranes. The interstitial cells vary considerably in their cytoplasmic density. The majority are quite light, but some appear extremely dense, and in addition usually have a more irregular cell surface, with numerous small pseudopodia. These differences may well reflect variations in physiological state. Cytoplasmic structures previously interpreted as "crystalloids" consist of long bundles of minute parallel tubules, each about 180 A in diameter, which seem to be local differentiations of the endoplasmic reticulum. The mitochondria are rod-shaped, and contain a moderately complex internal membrane structure, and also occasional large inclusions that are spherical and homogeneous. The prominent juxtanuclear Golgi complex contains closely packed flattened sacs and small vesicles. The results of the present study, coupled with biochemical evidence from other laboratories, make it seem highly probable that the agranular endoplasmic reticulum is involved in the synthesis of the steroid hormones produced by the interstitial cell. This finding therefore constitutes one of the first functions of the agranular reticulum for which there is good morphological and biochemical evidence.     

Irradiation of cells in tissue culture

Summary Cultures of human amnion were employed to check the hypothesis that cell strains with heteroploid chromosome counts regularly produce giant cells within 12 days following treatment with 2000 r and 4000 r of gamma irradiation from a cobalt source, while this response has not been obtained from primary cultures whose cells were presumed to be diploid.The giant cell reaction not only was obtained from two transfer passage lines of a well-established amnion strain developed at Berkeley (No A 185-21C-26 and No A 185-21C-45) but was also found for a 20-day second passage culture of amnion. Since this line has continued to reproduce at a rapid rate, it is presumed to have assumed the features of a typical strain within the period of observation. This impression was reinforced by the finding that the chromosome number for 32 cells fixed on the 35th day had a modal value of 67.In contrast, both untrypsinized and trypsinized spindle cells in primary cultures as well as unaltered epithelial elements which had not been subcultured gave no evidence of giant cell formation 12 days after exposure to 2000 r and 4000 r from a Cobalt⁶⁰ source.These data lend evidence that giant cell formation is related to the chromosomal constitution of the irradiated elements.This research was supported by funds provided under Contract AF 18(600)-1263 with the School of Aviation Medicine, USAF, Randolph Air Force Base, Texas.Fellow of the Instituto de Alta Cultura and the Fundacão Calouste Gulbenkian of Lisbon, Portugal.Tobacco Industry Research Committee Fellow.     

A first-generation map of the turkey genome.

A primary linkage map of the domestic turkey (Meleagris gallopavo) was developed by segregation analysis of genetic markers within a backcross family. This reference family includes 84 offspring from one F1 sire mated to two dams. Genomic DNA was digested using one of five restriction enzymes, and restriction fragment length polymorphisms were detected on Southern blots using probes prepared from 135 random clones isolated from a whole-embryo cDNA library. DNA sequence was subsequently determined for 114 of these cDNA clones. Sequence comparisons were done using BLAST searches of the GenBank database, and redundant sequences were eliminated. High similarity was found between 23% of the turkey sequences and mRNA sequences reported for the chicken. The current map, based on expressed genes, includes 138 loci, encompassing 113 loci arranged into 22 linkage groups and an additional 25 loci that remain unlinked. The average distance between linked markers is 6 cM and the longest linkage group (17 loci) measures 131 cM. The total map distance contained within linkage groups is 651 cM. The present map provides an important framework for future genome mapping in the turkey.     

Structure and properties of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) produced by Alcaligenes latus

Summary Random copolymers of 3-hydroxybutyrate (3HB) and 4-hydroxybutyrate (4HB) with a wide range of compositions varying from 0 to 83 mol% 4HB were produced by Alcaligenes latus from the mixed carbon substrates of 3-hydroxybutyric and 4-hydroxybutyric acids. The structure and physical properties of P(3HB-co-4HB) were characterized by¹H and¹³C NMR spectroscopy, gel-permeation chromatography, and differential scanning calorimetry. The isothermal radial growth rates of spherulites of P(3HB-co-4HB) were much slower than the rate of P(3HB) homopolymer. The enzymatic degradation rates of P(3HB-co-4HB) films by a PHB depolymerase were strongly influenced by the copolymer composition.     

The hyperthermophilic archaeon Pyrodictium occultum has two alpha-like DNA polymerases.

We cloned two genes encoding DNA polymerases from the hyperthermophilic archaeon Pyrodictium occultum. The deduced primary structures of the two gene products have several amino acid sequences which are conserved in the alpha-like (family B) DNA polymerases. Both genes were expressed in Escherichia coli, and highly purified gene products, DNA polymerases I and II (pol I and pol II), were biochemically characterized. Both DNA polymerase activities were heat stable, but only pol II was sensitive to aphidicolin. Both pol I and pol II have associated 5'-->3' and 3'-->5' exonuclease activities. In addition, these DNA polymerases have higher affinity to single-primed single-stranded DNA than to activated DNA; even their primer extension abilities by themselves were very weak. A comparison of the complete amino acid sequences of pol I and pol II with two alpha-like DNA polymerases from yeast cells showed that both pol I and pol II were more similar to yeast DNA polymerase III (ypol III) than to yeast DNA polymerase II (ypol II), in particular in the regions from exo II to exo III and from motif A to motif C. However, comparisons region by region of each polymerase showed that pol I was similar to ypol II and pol II was similar to ypol III from motif C to the C terminus. In contrast, pol I and pol II were similar to ypol III and ypol II, respectively, in the region from exo III to motif A. These findings suggest that both enzymes from P. occultum play a role in the replication of the genomic DNA of this organism and, furthermore, that the study of DNA replication in this thermophilic archaeon may lead to an understanding of the prototypical mechanism of eukaryotic DNA replication.     

Enhancement of aromatic amino acid-nucleic acid base stacking interaction by metal coordination to base: fluorescence study on a tryptophan-Pt(II)-guanine ternary complex

In order to investigate the effect of the Pt(II) ion on the stacking interaction between tryptophan and a guanine base, the quenching of Trp fluorescence was monitored for some systems in the absence and presence of the metal ion, and the association constants were obtained by the analysis of Eadie-Hofstee plots. All spectral data suggested that the stacking interaction is enhanced by the Pt(II) coordination to the guanine N7 atom. The result indicates the importance of the metal ion as a bookmark in the specific recognition of a nucleic acid base by an aromatic amino acid residue.