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.     

Measurement of cytosolic free magnesium ion concentration by 19F NMR

Fluorinated derivatives of the chelator o-aminophenol-N,N,O-triacetic acid (APTRA) have been developed, synthesized, and analyzed for use as 19F NMR indicators of free cytosolic magnesium concentration. Magnesium dissociation constants for the 4-fluoro, 5-fluoro, and 4-methyl-5-fluoro species were determined to be 3.1, 0.9, and 0.6 mM, respectively, on the basis of UV absorption measurements at 37 degrees C in 115 mM KCl and 20 mM NaCl, pH 7.1, buffered with 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid-tris-(hydroxymethyl)aminomethane. The corresponding pK values, which reflect protonation of the nitrogen atom, were determined by 19F NMR to be 4.15, 5.45, and 5.55, respectively, so that the chelators are insensitive to pH variations near the normal physiological range. The dissociation constants of these chelators for calcium ions are lower than those for magnesium but roughly 2-3 orders of magnitude above typical basal cytosolic free calcium levels, so that calcium ions will not interfere with the determinations of magnesium levels. 19F NMR studies carried out at 339.7 MHz indicate that magnesium ions are in slow exchange with the 5-fluoro and 4-methyl-5-fluoro APTRA derivatives and in fast exchange with the 4-fluoro APTRA derivative. In contrast, calcium ions were found to be in intermediate to fast exchange with all chelators. The apparent anomaly of higher thermodynamic stability of the APTRA complexes for calcium relative to magnesium but lower kinetic stability (higher k-1 values) for the calcium complexes reflects the very different association rates for the two ions. Thus, the magnesium association rates are 3 orders of magnitude slower than those for calcium ions.(ABSTRACT TRUNCATED AT 250 WORDS)     

13C NMR of the bases of three DNA oligonucleotide duplexes: assignment methods and structural features

Natural abundance 13C NMR spectra of three DNA oligomers have been obtained. Most of the base resonances are well resolved from one another. A combination of two independent methods was used in making assignments: a one-dimensional spectral comparison method and a two-dimensional proton-detected 1H-13C correlated experiment for the protonated carbons. There are large shielding changes (between 1.62 and -1.40 ppm) upon thermal dissociation of the duplex. The shapes of the chemical shift vs temperature curves are largely independent of sequence. The base carbon resonance frequencies are sensitive to hydrogen bonding, base stacking, sugar conformation, and changes in the glycosyl torsion angle.     

A phenotypic and functional analysis of long-lived B and T lymphocytes

The lymphocyte composition of spleen, lymph nodes, bone marrow, and thymus of mice submitted to hydroxyurea treatments for four consecutive days was studied. The treatment selects for small lymphocyte populations that represent between 4 and 20% of control numbers in the various organs. Spleen and bone marrow contain the same B cell population with a low IgM, high IgD, low I-E phenotype, which respond to LPS at control clonal frequencies. The T cell compartment is equally depleted, and the lymphocytes remaining contain frequencies of clonable cells in response to mitogens and IL-2 that are comparable to those detected in normal spleen cells. Overall, the results suggest that only a minor fraction of all lymphocytes in a normal young adult mouse have life spans longer than 4 days.     

Identification of the hepatocyte Na+-dependent bile acid transport protein using monoclonal antibodies

Monoclonal antibodies have been utilized to characterize the hepatocyte Na+-dependent bile acid transport system. Sinusoidal plasma membrane proteins in the 49-54-kDa range, which are thought to be components of this transport system, based on photo-affinity labeling and reconstitution studies, have been partially purified by affinity chromatography and utilized as an immunogen for the production of a panel of monoclonal antibodies (mAb). One of these mAbs, 25A-3, recognized both a 49- and a 54-kDa protein as assessed by immunoprecipitation. In addition, it was shown to protect the bile acid transport system from inhibition by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) in a dose-dependent manner. DIDS covalently labeled membrane proteins of 49 and 54 kDa, and this process could be significantly inhibited when performed in the presence of mAb 25A-3. Furthermore, the DIDS-labeled membrane proteins were immunoprecipitated by 25A-3. These results establish that one of these membrane components is the bile acid carrier protein. Another mAb (25D-1) which immunoprecipitated only a 49-kDa protein was shown to block the protective effect of 25A-3 on DIDS inhibition of bile acid transport. In addition both antibodies effected each other's binding capacity to hepatocytes and reacted with the same 49-kDa protein as established by sequential immunoprecipitation. Binding studies indicated that there are approximately 3.3 X 10(6) 49-kDa transport molecules/hepatocyte. These results firmly establish that the 49-kDa protein is the Na+-dependent hepatocyte bile acid transporter.     

Purification and reconstitution of the bile acid transport system from hepatocyte sinusoidal plasma membranes

The taurocholic acid transport system from hepatocyte sinusoidal plasma membranes has been studied using proteoliposome reconstitution procedures. Membrane proteins were initially solubilized in Triton X-100. Following detergent removal, the resultant proteins were incorporated into lipid vesicles prepared from soybean phospholipids (asolectin) using sonication and freeze-thaw procedures. The resultant proteoliposomes demonstrated Na+-dependent transport of taurocholic acid which could be inhibited by bile acids. Greatly reduced amounts of taurocholic acid were associated with the phospholipid or membrane proteins alone prior to proteoliposome formation. Membrane proteins were fractionated on an anionic glycocholate-Sepharose 4B affinity column which was prepared by coupling (3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholan-24-oyl)-N alpha-lysine to activated CH-Sepharose 4B via the epsilon-amino group of lysine resulting in the retention of a free carboxyl group. The adsorbed proteins enriched in components in the 54 kDa zone, which were originally identified by photoaffinity labeling to be components of the bile acid transport system, were also incorporated into liposomes. This vesicle system showed almost a 4-fold increase in Na+-dependent taurocholic acid uptake when compared to proteoliposomes formed from total membrane protein, as well as sensitivity to inhibition by bile acids. These results demonstrate that the bile acid carrier system can be reconstituted in proteoliposomes and that utilizing proteins in the 54 kDa zone leads to a significant enhancement in the transport capacity of the reconstituted system, consistent with the role of 54 kDa protein(s) as component(s) of the bile acid carrier system.     

Ultrastructure of the frontal organ in Convoluta and Macrostomum spp.: significance for models of the turbellarian archetype

Present models of turbellarian evolution depict the organism with a frontal organ — a complex of glands whose necks emerge at the anterior tip of the body — and therefore imply that this organ is homologous throughout the Turbellaria. However, comparisons of representatives of the Acoela and Macrostomida, two putatively primitive orders of the Turbellaria, show that frontal organs in these two are not similar in ultrastructure or histochemistry. The acoel Convoluta pulchra had a prominent cluster of frontal mucous glands whose necks emerged together in a frontal pore at the exact apical pole of the organism, and an array of smaller glands of at least five other types opened at the anterior end, separately from and ventral to this pore. The frontal organs (Stirndrüsen) of two species of Macrostomum on the other hand, comprised an array of discretely emerging necks of at least two gland types including one with rhabdiform (rhammite) and one with globular mucous secretion granules neither of which emerge at the apical pole. In neither species did the organ appear to be sensory. Our findings indicate a low probability of homology between the frontal glands of the Acoela and Macrostomida.     

Angiotensin II generated by a human renal carboxypeptidase

Angiotensin II, the potent hypertensive octapeptide, can be generated by a sequential cleavage of the carboxyl-terminal leucine and histidine from angiotensin I by a human renal extract. This extract does not hydrolyze further the resulting octapeptide. The more widely recognized biosynthetic pathway is by the extracellular dipeptide cleavage of angiotensin I by an enzyme which also degrades bradykinin, i.e., angiotensin converting enzyme. The presence of a carboxypeptidase activity capable of generating but not further hydrolyzing angiotensin II was observed in an ammonium sulfate fraction of a human renal extract. This novel enzymatic activity is distinct from angiotensin converting enzyme activity in that it is not dependent upon calcium and is not inhibited by known angiotensin converting enzyme inhibitors.     

Isolation of functional polyclonal TsF from MRL-lpr spleens using monoclonal antibody absorbance

We have previously described a monoclonal antibody, B16G, which has been found to be specific for T-cell derived suppressor factors (TsF). B16G has been shown to react with T-suppressor cells, TsF in the spleens of normal or tumor-bearing mice, the TsF produced by a tumor-specific T-cell hybridoma, and with polyclonal whole human TsF isolated from tonsilar tissue. This panreactivity inherent to the B16G MAb has made it clear that it recognizes some common, shared epitope of the TsF molecule. In this study we have used B16G as a probe to isolate TsF from the spleens of MRL-lpr mice and compare the activity with these factors isolated from the spleens of an MHC compatible nonautoimmune strain, CBA. We find that equivalent quantities of functional TsF are isolable from both strains and thus, it can be concluded that the associated oligoclonal B-cell activation characteristic of MRL-lpr mice is not due to a polyclonal T-suppressor cell deficit, nor to the ability of TsC in these mice to produce soluble, functional TsFs. The molecular and biochemical characteristics of these TsFs are discussed.     

Synergistic antitumor effect of interferon and anti-idiotype monoclonal antibody in murine lymphoma

Both IFN-alpha and anti-idiotype monoclonal antibody therapy have significant antitumor activity in vivo in a murine B cell lymphoma model. Combination therapy with syngeneic anti-idiotype antibody of the IgG2a or IgG2b isotype (a single i.p. injection of 100 micrograms) and recombinant human hybrid interferon-alpha A/D (10(4) to 10(6) U three times weekly for 3 wk) synergistically increased median survival time in mice challenged with a lethal dose of tumor cells compared with the sum of the median survival times of the two individual treatments. IFN-alpha has direct antiproliferative activity against 38C13 in vitro and enhances in vitro macrophage anti-idiotype antibody-specific cytolysis for IgG2a, IgG2b, and IgG1 isotypes.