Degradation of ethylene glycol and polyethylene glycols by methanogenic consortia.

Methanogenic enrichments capable of degrading polyethylene glycol and ethylene glycol were obtained from sewage sludge. Ethanol, acetate, methane, and (in the case of polyethylene glycols) ethylene glycol were detected as products. The sequence of product formation suggested that the ethylene oxide unit [HO-(CH2-CH2-O-)xH] was dismutated to acetate and ethanol; ethanol was subsequently oxidized to acetate by a syntrophic association that produced methane. The rates of degradation for ethylene, diethylene, and polyethylene glycol with molecular weights of 400, 1,000, and 20,000, respectively, were inversely related to the number of ethylene oxide monomers per molecule and ranged from 0.84 to 0.13 mM ethylene oxide units degraded per h. The enrichments were shown to best metabolize glycols close to the molecular weight of the substrate on which they were enriched. The anaerobic degradation of polyethylene glycol (molecular weight, 20,000) may be important in the light of the general resistance of polyethylene glycols to aerobic degradation.     

Recognition of Leishmania donovani antigens by murine T lymphocyte lines and clones. Species cross-reactivity, functional correlates of cell-mediated immunity, and antigen characterization

Studies in man and experimental animals suggest that cell-mediated immunity is of primary importance in limiting the pathogenesis of cutaneous and visceral leishmaniasis. In an attempt to determine, more directly, the role of T lymphocytes and the nature of the antigens that activate them, we have propagated antigen-specific murine T lymphocyte lines and clones that proliferate in response to antigens present on the membrane of intact Leishmania donovani promastigotes. One such line cross-reacts with membrane antigens on seven other Leishmania species and, to a lesser extent, with antigens on African procyclic trypanosomes. T lymphocyte clones that also exhibited a broad range of species cross-reactivity were isolated. About 40% of these clones had highly restricted specificity, whereas 60% were more extensively cross-reactive. The parent line and some clones passively transferred footpad DTH when injected locally, and some secreted a lymphokine activity that elicited intracellular killing of amastigotes within infected macrophages. Although the proliferative response of most clones was H-2 restricted, two clones appeared to be reactive in the presence of allogeneic antigen presenting cells. The majority of the clones appeared to recognize carbohydrate containing antigens, and absorption with solid substrate-bound lectins indicated that these antigens contained both mannose and galactose ligands. The antigenic activity was also absorbed using either of two extensively cross-reactive anti-parasite monoclonal antibodies.     

Production of Highly Labeled Adenoviruses

A method is described for increasing the incorporation of radioactive thymidine into adenovirus deoxyribonucleic acid by the use of amethopterin. In addition, a modified procedure is presented for the preparation of highly purified adenoviruses. This procedure, which employs enzymatic digestion of cellular debris, obviates the necessity for fluorocarbon treatment of crude virus suspensions, and routinely provides excellent recovery of virus.     

Cadmium evokes inositol polyphosphate formation and calcium mobilization. Evidence for a cell surface receptor that cadmium stimulates and zinc antagonizes

Cd2+ and other divalent metals mobilized cell Ca2+ in human skin fibroblasts. The divalent metals produced a large spike in cytosolic free Ca2+ and strikingly increased net Ca2+ efflux similarly to bradykinin. One-tenth microM Cd2+ half-maximally increased 45Ca2+ efflux. The potency order of the Ca2+ mobilizing metals was: Cd2+ greater than Co2+ greater than Ni2+ greater than Fe2+ greater than Mn2+. Cd2+ probably acts at an extracellular site because loading the cells with a heavy metal chelator only slightly inhibited Cd2+-evoked 45Ca2+ efflux. Cd2+ increased [3H]inositol polyphosphates; [3H]inositol trisphosphate increased 4-fold in 15 s. Zn2+ reversibly blocked 45Ca2+ efflux evoked by Cd2+ but not that produced by bradykinin. Zn2+ competitively (Ki = approximately 0.4 microM) inhibited net Ca2+ efflux produced by Cd2+. Cd2+ also evoked Ca2+ mobilization in umbilical artery muscle, endothelial, and neuroblastoma cells, and the divalent cation agonist and antagonist specificities were similar to those in the fibroblasts. The divalent metals appear to trigger Ca2+ mobilization via a reversible interaction with an external site on the cell surface, which may be considered a "Cd2+ receptor."     

Expression and transfer of engineered catabolic pathways harbored by Pseudomonas spp. introduced into activated sludge microcosms.

Two genetically engineered microorganisms (GEMs), Pseudomonas sp. strain B13 FR1(pFRC20P) (FR120) and Pseudomonas putida KT2440(pWWO-EB62) (EB62), were introduced into activated sludge microcosms that had the level of aeration, nutrient makeup, and microbial community structure of activated sludge reactors. FR120 contains an experimentally assembled ortho cleavage route for simultaneous degradation of 3-chlorobenzoate (3CB) and 4-methyl benzoate (4MB); EB62 contains a derivative TOL plasmid-encoded degradative pathway for toluene experimentally evolved so that it additionally processes 4-ethyl benzoate (4EB). Experiments assessed survival of the GEMs, their ability to degrade target substrates, and lateral transfer of plasmid-encoded recombinant DNA. GEMs added at initial densities of 10(6) to 10(7) bacteria per ml of activated sludge declined to stable population densities of 10(4) to 10(5) bacteria per ml. FR120 degraded combinations of 3CB and 4MB (1 mM each) following 3 days of adaptation in the microcosms. Indigenous microorganisms required an 8-day adaptation period before degradation of 4MB was observed; 3CB was degraded only after the concentration of 4MB was much reduced. The indigenous microbial community was killed when both compounds were present at concentrations of 4.0 mM. However, in parallel microcosms containing FR120, the microbial community maintained a normal density of viable cells. Indigenous microbes readily degraded 4EB (2 mM), and EB62 did not significantly increase the observed rate of degradation. In filter matings, transfer of pFRC20P, which specifies mobilization but not transfer functions, from FR120 to P. putida UWC1 was not detectable (< 10(-7) transconjugants per donor cell).(ABSTRACT TRUNCATED AT 250 WORDS)     

Survival and function of a genetically engineered Pseudomonad in aquatic sediment microcosms.

Pseudomonas sp. strain B13 FR1(pFRC20P) is a genetically engineered microorganism (GEM) which is able to degrade chloro- and methylaromatics through a constructed ortho cleavage pathway. The fate of the GEM and its ability to degrade substituted aromatic compounds in two different aquatic sediments was investigated by using a microcosm system which consisted of intact layered sediment cores with an overlying water column. The GEM survived in Lake Plussee and in Rhine river sediments at densities of approximately 10(5) bacteria per g (dry weight) (1 to 5% of the total CFU) throughout a 4-week period of investigation. According to several criteria, the microcosm system was stable and healthy throughout the experiment and the addition of the GEM did not affect the total number of extractable CFU (I. Wagner-Döbler, R. Pipke, K. N. Timmis, and D. F. Dwyer, Appl. Environ. Microbiol. 58:1249-1258, 1992). When compared with uninoculated controls, the presence of the GEM enhanced the rate of degradation of a mixture of 3-chlorobenzoate and 4-methylbenzoate (25 microns each) which had been added to the water column of the sediment cores.     

Brassica S-Proteins Accumulate in the Intercellular Matrix along the Path of Pollen Tubes in Transgenic Tobacco Pistils

A tobacco plant transformed with a Brassica oleracea SLG-22 gene was analyzed by immunocytochemical methods to determine the localization of the transgene-encoded protein product. Immunolabeling was observed in the pistil along the path followed by pollen tubes after pollination. S-antigen accumulated in the intercellular matrix of the transmitting tissue of the style and its continuation in the basal portion of the stigma and outside a few special cells of the placental epidermis of the ovary. This pattern of S-antigen distribution closely resembles that described for the S-associated glycoproteins of self-incompatible Nicotiana alata and differs from its distribution in B. oleracea.     

Antisense oligonucleotide containing an internal, non-nucleotide-based linker promote site-specific cleavage of RNA.

We have designed and synthesized a series of novel antisense methylphosphonate oligonucleotide (MPO) cleaving agents that promote site-specific cleavage on a complementary RNA target. These MPOs contain a non- nucleotide-based linking moiety near the middle of the sequence in place of one of the nucleotide bases. The region surrounding the unpaired base on the RNA strand (i.e. the one directly opposite the non-nucleotide-linker) is sensitive to hydrolytic cleavage catalyzed by ethylenediamine hydrochloride. Furthermore, the regions of the RNA comprising hydrogen bonded domains are resistant to cleavage compared with single-stranded RNA alone. Several catalytic moieties capable of supporting acid/base hydrolysis were coupled to the non-nucleotide-based linker via simple aqueous coupling chemistries. When tethered to the MPO in this manner these moieties are shown to catalyze site-specific cleavage on the RNA target without any additional catalyst.     

Calcium mobilization and muscle contraction induced by acetylcholine in swine trachealis

The roles of Ca²⁺ mobilization in development of tension induced by acetylcholine (ACh, 0.1–100 µM) in swine tracheal smooth muscle strips were studied. Under control conditions, ACh induced a transient increase in free cytosolic calcium concentration ([Ca²⁺]_i) that declined to a steady-state level. The peak increase in [Ca²⁺]_i correlated with the magnitude of tension at each [ACh] after a single exposure to ACh, while the steady-state [Ca²⁺]_i did not. Removal of extracellular Ca²⁺ had little effect on peak [Ca²⁺]_i but greatly reduced steady-state increases in [Ca²⁺]_i and tension. Verapamil inhibited steady-state [Ca²⁺]_i only at [ACh]<1 µM. After depletion of internal Ca²⁺ stores by 10 min exposure to ACh in Ca²⁺-free solution and then washout of ACh for 5 min in Ca²⁺-free solution, simultaneous re-exposure to ACh in the presence of 2.5 mM Ca²⁺ increased [Ca²⁺]_i to the control steady-state level without overshoot. The tension attained was the same as control for each [ACh] used. Continuous exposure to successively increasing [ACh] (0.1–100 µM) also reduced the overshoot of [Ca²⁺]_i at 10 and 100 µM ACh, yet tension reached control levels at each [ACh] used. We conclude that the steady-state increase in [Ca²⁺]_i is necessary for tension maintenance and is dependent on Ca²⁺ influx through voltage-gated calcium channels at 0.1 µM ACh and through a verapamil-insensitive pathway at 10 and 100 µM. The initial transient increase in calcium arises from intracellular stores and is correlated with the magnitude of tension only in muscles that have completely recovered from previous exposure to agonists.     

Brownian dynamics simulations of probe and self-diffusion in concentrated protein and DNA solutions.

We have developed a Brownian dynamics algorithm for simulating probe and self-diffusion in concentrated solutions of DNA and protein. In these simulations, proteins are represented as spheres with radii given by their hydrodynamic radii, while DNA is modeled as a wormlike chain of hydrodynamically equivalent spherical frictional elements. The molecular interaction potentials employed by the program allow for intramolecular stretching and bending motions of the DNA chains, short-range Lennard-Jones interactions, and long-range electrostatic interactions. To test the program, we have carried out simulations of bovine serum albumin (BSA) probe diffusion and DNA self-diffusion in solutions of short-chain DNA as a function of both DNA concentration and solution ionic strength. In addition, we report on simulations of BSA self-diffusion as a function of BSA concentration and ionic strength. Based on a comparison to available experimental data, we find that our simulations accurately predict these transport properties under conditions of physiological salt concentration and predict the stronger concentration dependence observed at lower salt concentrations. These results are discussed in light of the nature of the intermolecular interactions in such systems and the approximations and limitations of the simulation algorithm.