Survival of Escherichia coli with and without ColE1::Tn5 after aerosol dispersal in a laboratory and a farm environment.

The survival of a laboratory strain and a naturally occurring fecal strain of Escherichia coli, with and without a Tn5-containing derivative of ColE1, was examined after aerosol dispersal in a laboratory office and a barn under ambient temperature and humidity conditions. Following the release of paired strains, air and diverse types of surfaces were assayed for the test organisms. In both environments, the number of airborne bacteria declined rapidly within the first 2 h. Longer survival was found on surfaces and varied with surface type: recovery was greatest from wood products. Organisms persisted for 1 day in the office and for up to 20 days in the barn. Survival of the fecal strain was better than that of the laboratory strain in both test environments. In general, plasmid-bearing strains fared similarly to their plasmidless parents, but in several comparisons the ColE1::Tn5-containing strain showed enhanced survival. These studies have implications for the present and proposed release of genetically engineered organisms with and without plasmid vectors.     

Characterization of a developmentally transient adrenergic depolarizing response in cultured locus coeruleus neurons

The effects of iontophoretically applied noradrenaline have been tested on intracellularly recorded locus coeruleus neurons grown in explant cultures from neonatal mice. In addition to hyperpolarizing responses mediated by alpha 2-adrenergic receptors, as observed in locus coeruleus neurons in vivo and in brain slices from adult animals, alpha 1-mediated depolarizations were observed to succeed the initial hyperpolarizations in some cultures. It was shown that the depolarizing responses were only present in younger cultures, i.e., less than 26 days in vitro. In cultures less than 20 days old, all cells displayed the biphasic hyperpolarizing-depolarizing responses. Both components of the response appear to be direct, since they were present when synaptic transmission was blocked by including tetrodotoxin or by altering divalent cations in the perfusate. The depolarizing responses were frequently reduced in solutions with altered divalent cation content, and this might reflect a calcium dependency of this response. The hyperpolarizing and depolarizing components of the responses to noradrenaline were progressively blocked by increasing concentrations of the selective antagonists yohimbine and prazosin, respectively, in the dose ranges of 100 mM - 1 microM (yohimbine) and 20-200 nM (prazosin). Recent results from electrophysiological studies of locus coeruleus neurons in brain slices suggest that similar changes occur in the animal as well as in culture. It is possible that the transient depolarizing responses reflect a developmentally important enhanced responsiveness of locus coeruleus neurons during the early postnatal period.     

Identification and assignment of base pairs in three helical stems of Torulopsis utilis ribosomal 5S RNA and its RNase T1 and RNase T2 cleaved fragments via 500-MHz proton homonuclear overhauser enhancements

Imino proton resonances in the downfield region (10-14 ppm) of the 500-MHz 1H NMR spectrum of Torulopsis utilis 5S RNA are identified (A X U, G X C, or G X U) and assigned to base pairs in helices I, IV, and V via analysis of homonuclear Overhauser enhancements (NOE) from intact T. utilis 5S RNA, its RNase T1 and RNase T2 digested fragments, and a second yeast (Saccharomyces cerevisiae) 5S RNA whose nucleotide sequence differs at only six residues from that of T. utilis 5S RNA. The near-identical chemical shifts and NOE behavior of most of the common peaks from these four RNAs strongly suggest that helices I, IV, and V retain the same conformation after RNase digestion and that both T. utilis and S. cerevisiae 5S RNAs share a common secondary and tertiary structure. Of the four G X U base pairs identified in the intact 5S RNA, two are assigned to the terminal stem (helix I) and the other two to helices IV and V. Seven of the nine base pairs of the terminal stem have been assigned. Our experimental demonstration of a G X U base pair in helix V supports the 5S RNA secondary structural model of Luehrsen and Fox [Luehrsen, K. R., & Fox, G.E. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 2150-2154]. Finally, the base-pair proton peak assigned to the terminal G X U in helix V of the RNase T2 cleaved fragment is shifted downfield from that in the intact 5S RNA, suggesting that helices I and V may be coaxial in intact T. utilis 5S RNA.     

Alginate Production by Plant-Pathogenic Pseudomonads

Eighteen plant-pathogenic and three non-plant-pathogenic pseudomonads were tested for the ability to produce alginic acid as an exopolysaccharide in vitro. Alginate production was demonstrated for 10 of 13 fluorescent plant-pathogenic pseudomonads tested with glucose or gluconate as the carbon source, but not for all 5 nonfluorescent plant pathogens and all 3 non-plant pathogens tested. With sucrose as the carbon source, some strains produced alginate while others produced both polyfructan (levan) and alginate. Alginates ranged from <1 to 28% guluronic acid, were acetylated, and had number-average molecular weights of 11.3 × 10³ to 47.1 × 10³. Polyfructans and alginates were not elicitors of the soybean phytoalexin glyceollin when applied to wounded cotyledon surfaces and did not induce prolonged water soaking of soybean leaf tissues. All or most pseudomonads in rRNA-DNA homology group I may be capable of synthesizing alginate as an exopolysaccharide.     

Demonstration of the GC-rich common arm in yeast ribosomal 5.8S RNA via 500-MHz proton nuclear magnetic resonance and Overhauser enhancements

In this paper we report the first 1H NMR study of the base-paired secondary structure of yeast 5.8S RNA. On the basis of a combination of homonuclear Overhauser enhancements and temperature dependence of the proton 500-MHz NMR spectrum, we are able to identify and assign eight of the nine base pairs in the most thermally stable helical arm: G116.C137-C117.G136-C118.G135- C119.G134-C120.G133-U121.G132- U122.A131-G123.C130. This arm contains an unusually temperature-stable (to 71 degrees C) segment of four consecutive G.C base pairs. This work constitutes the most direct evidence to date for the existence and base-pair sequence of the GC-rich helix, which is common to most currently popular secondary structural models for eukaryotic 5.8S ribosomal RNA.     

Regulation of intracellular pH in LLC-PK1 cells by Na+/H+ exchange

Summary Suspensions of LLC-PK₁ cells (a continuous epitheliod cell line with renal characteristics) are examined for mechanisms of intracellular pH regulation using the fluorescent probe BCECF. Initial experiments determine suitable calibration procedures for use of the BCECF fluorescent signal. They also determine that the cell suspension contains cells which (after 4 hr in suspension) have Na⁺ and K⁺ gradients comparable to those of cells in monolayer culture. The steady-state intracellular pH (7.05±0.01,n=5) of cells which have recovered in (pH 7.4) Na⁺-containing medium is not affected over several minutes by addition of 100 M amiloride or removal of extracellular Na⁺ (Na _o ⁺ /H _i ⁺ and Na _i ⁺ /H _o ⁺ exchange reactions are functionally inactive (compared to cellular buffering capacity). In contrast, Na _o ⁺ /H _i ⁺ exchange is activated by an increased cellular acid load. This activation may be observed directly either as a stimulation of net H⁺ efflux or net Na⁺ influx with decreasing intracellular pH. The extrapolation of this latter data suggests a set point of Na⁺/H⁺ exchange of approximately pH 7.0, consistent with the observed resting intracellular pH of approximately 7.05.     

Characterization of envelope proteins of infectious bovine rhinotracheitis virus (bovine herpesvirus 1) by biochemical and immunological methods.

Ten glycoproteins of molecular weights of 180,000, 150,000, 130,000, 115,000, 97,000, 77,000, 74,000, 64,000, 55,000, and 45,000 (designated as 180K, 150K, etc.) and a single nonglycosylated 107,000-molecular-weight (107K) protein were quantitatively removed from purified bovine herpesvirus 1 (BHV-1) virions by detergent treatment. Immunoprecipitations with monospecific and monoclonal antibodies showed that three sets of coprecipitating glycoproteins, 180K/97K, 150K/77K, and 130K/74K/55K, were the major components of the BHV-1 envelope. These glycoproteins were present in the envelope of the virion and on the surface of BHV-1-infected cells and reacted with neutralizing monoclonal and monospecific antibodies. Antibodies to 150K/77K protein had the largest proportion of virus-neutralizing antibodies, followed by antibodies to 180K/97K protein. Monoclonal antibodies to 130K/74K/55K protein were neutralizing but only in the presence of complement; however, monospecific antisera produced with 55K protein did not have neutralizing activity. Analysis under nonreducing conditions showed that the 74K and 55K proteins interact through disulfide bonds to form the 130K molecule. Partial proteolysis studies showed that the 180K protein was a dimeric form of the 97K protein and that the 150K protein was a dimer of the 77K protein, but these dimers were not linked by disulfide bonds. The 107K protein was not glycosylated and induced antibodies that did not neutralize BHV-1. The 64K protein was not precipitated by anti-BHV-1 convalescent antisera, and monospecific antisera to this protein precipitated several polypeptides from uninfected cell lysates, suggesting that 64K is a protein of cellular origin associated with the BHV-1 virion envelope.     

D-Ala6-des-Gly10-gonadotropin-releasing hormone ethylamide: absence of binding sites and lack of a direct effect in rabbit corpora lutea

Rat ovarian tissue has been shown to contain high-affinity gonadotropin-releasing hormone (GnRH) receptors, and synthetic GnRH analogues have been shown to inhibit steroid production by rat corpora lutea in vivo and in vitro. These results raise the possibility that an ovarian GnRH-like peptide may be involved in normal luteal regression. We have examined binding of D-Ala6-des-Gly10-GnRH ethylamide (D-Ala) to rabbit corpora lutea, and have investigated the luteolytic activity of this analogue in hypophysectomized, pseudopregnant rabbits. Three hypophysectomized estrogen-treated rabbits were injected with 0.25 mg D-Ala s.c. every 6 h for 48 h during mid-pseudopregnancy, and three were injected with vehicle only. Treatment with D-Ala produced no acute changes in serum progesterone, nor was the time of luteal regression altered. Rabbit anterior pituitary tissue was found to contain high-affinity GnRH receptors (Ka = 7.0 X 10(9) M-1; 188.2 +/- 35.6 fmol/mg protein). However, no similar high-affinity GnRH receptors were detected in rabbit luteal tissue from any stage of pseudopregnancy. Some apparent low-affinity binding was observed, but this displaceable binding was subsequently observed in all control tissues tested. Thus, a potent GnRH analogue does not have any detectable direct effect on steroidogenesis in the rabbit corpus luteum, nor are high-affinity GnRH binding sites present in rabbit luteal tissue.     

Attempts to probe the antigens and protective immunogens of Trichostrongylus colubriformis in immunoblots with sera from infected and hyperimmune sheep and high- and low-responder guinea pigs

Comparison of antisera from sheep during primary infection and following vaccination and challenge with Trichostrongylus colubriformis, with antisera obtained following primary infection of high- and low-responder guinea pigs, failed to reveal different antigenic patterns in proteins separated from fourth stage larval extracts by two-dimensional electrophoresis and probed by the immunoblot technique.Generally, serum IgG reacted specifically with worm antigens of mol. wt greater than 94,000, whereas protection against challenge infection was elicited most effectively in the guinea pig by fractions in the 67,000–94,000 range.Most distinct separations of larval proteins by SDS-polyacrylamide gel electrophoresis were obtained by extraction of live larvae and the extracts used within 2–3 days.