Steroid Lysis of Protoplasts and Effects of Stabilizers and Steroid Antagonists

Six synthetic antimicrobial steroids were examined for indications of their mechanism of action. Dequadin acetate, cetyl pyridinium chloride (CPC), and sodium deoxycholate were studied for comparison. Aerated cells of Sarcina lutea were washed, suspended in 1.06 M sucrose, and converted to protoplasts with 20 mug/ml of lysozyme. Lysis was measured optically at 650 mmu as a decrease in optical density. Screening tests with 50 mug/ml of each compound showed five steroids and CPC to be lytic. Protoplasts were strongly protected from lysis by pretreatment with 0.001 to 0.004 M spermine tetrahydrochloride. Other polyamines, such as spermidine phosphate, were less protective, and putrescine was ineffective. Uranyl nitrate (5 x 10(-4) M) rapidly agglutinated protoplasts and protected them from rupture by the lytic agents. Similar studies with 0.001 to 0.004 M Mg(++) showed varying degrees of protection, which, in most cases, was only temporary. Steroidal lysis did not appear to be related to chelation, since ethylenediaminetetraacetate did not cause lysis alone and antagonized some lytic compounds. Lecithin, Tween 80, Tween 20, and Span 20 at 0.05% exhibited certain effects on protoplast stability. Span 20 strongly prevented lysis by steroids. Tween 20 alone quickly caused protoplast rupture. Lecithin and Tween 80, which also caused lysis alone, interfered with lytic steroids and CPC. The test compounds were both inhibitory and lethal to cells of Sarcina lutea. The results suggest that direct action on cell membranes may be chiefly responsible for the antimicrobial properties of the steroids.     

Growth of epithelium from a preneoplastic mammary outgrowth in response to mammary adipose tissue

Summary We investigated the effects of conditioned media derived from mouse mammary fat pads on the proliferation of CL-S1 cells, an epithelial cell line originally isolated from a preneoplastic mammary outgrowth line. Cell proliferation in vitro in serum-free defined medium was compared to that in this medium conditioned using intact mammary fat pad pieces or isolated fat pad adipocytes. Culture medium was conditioned by incubating the conditioning material in defined culture medium for 24 h at 37°C. Conditioned medium induced CL-S1 proliferation as much as 10- to 20-fold above the minimal levels of growth in control cultures after 13 d of culture. The growth-stimulatory factor(s) had an apparent molecular weight of greater than 10 kDa. This growth-stimulatory activity was both heat and trypsin stable. Because the role of adipose tissue is to store and release lipids, we next tested whether lipids are released during medium conditioning. The lipid composition of the fat pad conditioned medium was characterized using both thin layer and gas liquid chromatography. These lipid analyses indicated that the fat pad pieces released significant amounts of fatty acids and phospholipids into the medium during the conditioning period. The free fatty acid composition included both saturated and unsaturated molecules, and about 80% of the total fatty acids consisted of palmitate, stearate, oleate, and linoleate. These same fatty acids were a structural component of the majority of phospholipid found in the medium. The addition of palmitate or stearate to defined medium had no effect or was inhibitory for CL-S1 proliferation, depending on the concentration used. Defined medium supplemented with oleate, arachidonate, or linoleate induced CL-S1 proliferation, and the inhibitory effects of palmitate and stearate were overcome by addition of oleate and linoleate. These data indicate that both unsaturated and saturated fatty acids are released from intact adipose cells of the mouse mammary fat pad and that fatty acids can influence the growth of prenoplastic mouse mammary epithelium. Thus, unsaturated fatty acids, perhaps in conjunction with other substances released simultaneously, are candidate molecules for the substances that mediate the effect of adipose tissue on growth of epithelium. This work was supported in part by a grant from the American Institute for Cancer Research; grant CA 46885 from the National Institutes of Health, Bethesda, MD; and by State of Washington initiative 171.     

Survey of equipment in general practice.

Partners in general practice have to buy any equipment they want themselves. As a result partners in high investing practices have lower net incomes. Of the 297 practices in Devon and Cornwall, 265 responded to a questionnaire listing 115 possible items of practice equipment. Overall, practices seemed to be fairly well equipped. Key findings were that 193 of those who responded had an electrocardiograph, 206 had a kit for minor operations, 119 owned a computer, and less than one third owned a microscope. Most of these practices were high investors. There seems to be a shift away from some traditional instruments towards expensive information technology. Government policies are encouraging the use of computers and such equipment, though funds are not necessarily being made available for this purpose.     

Gene expression in visceral endoderm: a comparison of mutant and wild- type F9 embryonal carcinoma cell differentiation

We have examined the abundance and cell specificity of several mRNAs that are regulated during the retinoic acid (RA)-induced differentiation of F9 embryonal carcinoma cells to visceral endoderm. The experiments confirmed the multistep nature of this process by demonstrating the expression of the ERA-1/Hox 1.6 message within 6 h after RA addition; the expression of messages specific for the extracellular matrix proteins laminin B1 and B2, and collagen IV(alpha 1) between days 4 and 12; and the expression of two visceral endoderm markers, alpha-fetoprotein (AFP) and H19, by days 8-15. In situ hybridization experiments revealed that the collagen IV(alpha 1) mRNA is restricted to the outer cell layer of F9 cell aggregates regardless of the presence or absence of RA. Laminin B1 and B2 mRNAs are concentrated in the outer cell layer of RA-treated aggregates although significant levels of message are also observed within the interior cells of the aggregates. Unexpectedly, AFP mRNA is detectable in only a subset of the outer cells of F9 cell aggregates grown 15 d in the presence of RA. The results obtained from wild-type F9 cells were compared with those from a mutant F9 cell line, RA-5-1, which was previously shown to synthesize collagen IV containing six- to ninefold less 4-hydroxyproline than that in wild-type F9 cells. RA-5-1 cells exhibit four- to sixfold less of the mRNAs encoding two visceral endoderm proteins, AFP and H19, than wild-type F9 cells after RA treatment of RA-5-1 aggregates. RA-5-1 cells, however, do exhibit an RA-associated increase in the level of ERA-1/Hox 1.6 mRNA within 6 h after adding RA. Although the collagen IV protein level is similar in wild-type F9 and RA-5-1 aggregates, the collagen IV(alpha 1) message level is 6-20-fold greater in aggregates of mutant cells than in aggregates of wild-type cells. Moreover, in situ hybridizations showed that this message is evenly distributed throughout the RA-5-1 aggregates rather than restricted to the outer cell layers as it is in wild-type F9 aggregates. These results suggest that abnormal collagen IV expression and localization are associated with decreased expression of the visceral endoderm markers, AFP and H19, in RA-5-1 cell aggregates.     

Studies of cloned simian immunodeficiency virus-specific T lymphocytes. gag-specific cytotoxic T lymphocytes exhibit a restricted epitope specificity.

CD8+ CTL inhibit the replication of HIV and simian immunodeficiency virus of macaques (SIVmac) in PBL and, therefore, are likely to play an important role in containing the spread of the AIDS virus in infected individuals. We have generated a series of gag-specific lytic T lymphocyte clones from PBL: of an SIVmac-infected rhesus monkey. These T cell clones are CD3+CD8+ and are MHC class I-restricted in their target specificity. They are, therefore, CTL. Interestingly, all gag-specific CTL clones, as well as the gag-specific lytic activity of PBL of this monkey, demonstrated specificity for a single 25 amino acid fragment of the SIVmac gag protein. Moreover, they were restricted in their lytic function by a single MHC class I allele. These findings illustrate a powerful method for cloning AIDS virus-specific T lymphocytes and demonstrate a remarkably restricted epitope specificity of this AIDS virus-specific CTL response.     

Molecular cloning of cDNA that encode MHC class I molecules from a New World primate (Saguinus oedipus). Natural selection acts at positions that may affect peptide presentation to T cells

To investigate the evolutionary pressures that drive the generation of polymorphism in primate MHC class I molecules, three cDNA that encode MHC class I alleles from a New World monkey, the cotton-top tamarin (Saguinus oedipus), were cloned and sequenced. These tamarin MHC class I alleles contained amino acid substitutions not found in any of the previously sequenced human MHC class I alleles. Moreover, the majority of these unique amino acid substitutions was located in the Ag recognition site at positions that have been shown to be critical in the presentation of viral peptides to T cells in mice and humans. These data suggest that selective pressures on MHC class I molecules preferentially act on the Ag recognition site and that the peptide binding or presenting functions of these molecules may drive the generation of MHC class I polymorphism. The novel Ag recognition sites of the tamarin MHC class I molecules, in addition to their restricted polymorphism, might account for the unusual susceptibility of the cotton-top tamarin to human pathogens.     

Detection of a specific isoform of alpha-actinin with antisera directed against dystrophin

We have characterized a protein immunologically related to dystrophin, the protein product of the Duchenne muscular dystrophy gene. We identify this related protein as a fast-twitch glycolytic isoform (mouse extensor digitorum longus-specific) of myofibrillar alpha-actinin. This specific isoform of alpha-actinin exhibits a more restricted pattern of expression in skeletal muscle than fast-twitch-specific isoforms of both myosin and Ca2+-ATPase. Our results provide evidence that dystrophin and myofibrillar alpha-actinin are related proteins, reinforcing the previous data concerning the sequence homologies noted between nonmuscle cytoskeletal alpha-actinin and dystrophin. In addition, we describe the first antisera directed against a specific myofibrillar skeletal muscle isoform of alpha-actinin.     

A genetic linkage map of human chromosome 21: analysis of recombination as a function of sex and age.

A genetic linkage map of human chromosome 21 has been constructed using 22 anonymous DNA markers and five complementary DNAs (cDNAs) encoding the amyloid beta protein precursor (APP), superoxide dismutase 1 (SOD1), the ets-2 proto-oncogene (ETS2), the estrogen inducible breast cancer locus (BCEI), and the leukocyte antigen, CD18 (CD18). Segregation of RFLPs detected by these DNA markers was traced in the Venezuelan Reference Pedigree (VRP). A comprehensive genetic linkage map consisting of the 27 DNA markers spans 102 cM on the long arm of chromosome 21. We have confirmed our initial findings of a dramatically increased rate of recombination at the telomere in both females and males and of significantly higher recombination in females in the pericentromeric region. By comparing patterns of recombination in specific regions of chromosome 21 with regard to both parental sex and age, we have now identified a statistically significant downward trend in the frequency of crossovers in the most telomeric portion of chromosome 21 with increasing maternal age. A less significant decrease in recombination with increasing maternal age was observed in the pericentromeric region of the chromosome. These results may help in ultimately understanding the physical relationship between recombination and nondisjunction in the occurrence of trisomy 21.