Structural gene for NAD synthetase in Salmonella typhimurium.

We have identified the structural gene for NAD synthetase, which catalyzes the final metabolic step in NAD biosynthesis. This gene, designated nadE, is located between gdh and nit at 27 min on the Salmonella typhimurium chromosome. Mutants of nadE include those with a temperature-sensitive lethal phenotype; these strains accumulate large internal pools of nicotinic acid adenine dinucleotide, the substrate for NAD synthetase. Native gel electrophoresis experiments suggest that NAD synthetase is a multimeric enzyme of at least two subunits and that subunits from Escherichia coli and S. typhimurium interact to form an active heteromultimer.     

IL-4, but not IL-5, can act synergistically with B cell activating factor (BCAF) to induce proliferation of resting B cells

B cell activating factor (BCAF) was initially identified in the supernatant of the murine T helper cell clone 52-3 (52-3 SN) because of its ability to promote activation and proliferation of resting B cells in the absence of any other costimulus. In this paper, we show that 52-3 T helper cells also secrete IL-4 and IL-5 and we have analyzed the influence of these two lymphokines on B cell proliferation induced by BCAF-containing 52-3 SN. Using the neutralizing anti-IL-4 monoclonal antibody 11B11, we observed partial inhibition of B cell proliferation. 52-3 SN free of IL-4 prepared using an immunoabsorbent column was still able to induce significant B cell proliferation. Although recombinant IL-4 alone does not induce B cell proliferation, it increased the proliferation induced by IL-4-free 52-3 SN. Kinetic studies showed that IL-4 is required at the start of B cell cultures in order to exert optimal synergistic effects. In contrast, anti-IL-5 monoclonal antibody NC17 did not affect the B cell proliferative activity of 52-3 SN whether or not IL-4 was present. When 52-3 SN was tested on dextran-sulfate-activated B cells, IL-5 and BCAF activities were detected but only the IL-5 activity was neutralized by monoclonal antibody NC17. These results demonstrate that (i) BCAF-containing SN can induce proliferation of resting B cells independently of IL-4 and IL-5, and (ii) IL-4, but not IL-5, can act synergistically with BCAF to induce B cell proliferation.     

Identification of mannose 6-phosphate in two asparagine-linked sugar chains of recombinant transforming growth factor-beta 1 precursor

Recombinant transforming growth factor-beta 1 (TGF-beta 1) precursor produced and secreted by a clone of Chinese hamster ovary cells was found to be glycosylated and phosphorylated. Treatment of 32P-labeled precursor protein with N-glycanase indicated that phosphate was incorporated into asparagine-linked complex carbohydrate moieties. Fractionation of 32P-labeled glycopeptides followed by amino acid sequence analysis indicated that greater than 95% of the label was incorporated into two out of three glycosylation sites at Asn-82 and Asn-136 of the TGF-beta 1 precursor. Two-dimensional electrophoretic analysis of acid hydrolyzed precursor protein and precursor protein-derived glycopeptides indicated that 32P was incorporated as mannose 6-phosphate. Binding studies with the purified receptor for mannose 6-phosphate indicated that the TGF-beta 1 precursor could bind to this receptor and the binding was specifically inhibited with mannose 6-phosphate.     

Activation of bovine neutrophils by recombinant interferon-gamma

The effect of recombinant bovine interferon-gamma (r-IFN-gamma) on neutrophil functions was investigated and compared to the effects of an unpurified lymphokine preparation. Incubation of purified bovine neutrophils with r-IFN-gamma or antigen-induced lymphokine for 2.5 hr at 37 degrees C resulted in impairment of the ability of neutrophils to migrate under agarose, and an enhancement of their ability to mediate antibody-dependent and antibody-independent cell-mediated cytotoxicity against chicken erythrocytes. Neither the lymphokine preparation nor the r-IFN-gamma had any influence on Staphylococcus aureus ingestion, or iodination by neutrophils. The lymphokine preparation enhanced cytochrome c reduction by neutrophils and was weakly chemotactic, whereas the r-IFN-gamma had neither of these effects. Only 5 min of r-IFN-gamma preincubation with neutrophils were needed to trigger protein synthesis by the neutrophils resulting in inhibition of random migration. Therefore, recombinant interferon-gamma acts as a neutrophil migration inhibition factor and a neutrophil activation factor resulting in enhanced neutrophil-mediated antibody-dependent and -independent cell-mediated cytotoxicity. Many, but not all, of the in vitro effects of an unpurified lymphokine preparation on neutrophil function can be attributed to the interferon-gamma contained in the lymphokine.     

Trimethyloxonium modification of single batrachotoxin-activated sodium channels in planar bilayers. Changes in unit conductance and in block by saxitoxin and calcium

Single batrachotoxin-activated sodium channels from rat brain were modified by trimethyloxonium (TMO) after incorporation in planar lipid bilayers. TMO modification eliminated saxitoxin (STX) sensitivity, reduced the single channel conductance by 37%, and reduced calcium block of inward sodium currents. These effects always occurred concomitantly, in an all-or-none fashion. Calcium and STX protected sodium channels from TMO modification with potencies similar to their affinities for block. Calcium inhibited STX binding to rat brain membrane vesicles and relieved toxin block of channels in bilayers, apparently by competing with STX for the toxin binding site. These results suggest that toxins, permeant cations, and blocking cations can interact with a common site on the sodium channel near the extracellular surface. It is likely that permeant cations transiently bind to this superficial site, as the first of several steps in passing inward through the channel.     

In situ hybridization at the electron microscope level: localization of transcripts on ultrathin sections of Lowicryl K4M-embedded tissue using biotinylated probes and protein A-gold complexes

A technique has been developed for localizing hybrids formed in situ on semi-thin and ultrathin sections of Lowicryl K4M-embedded tissue. Biotinylated dUTP (Bio-11-dUTP and/or Bio-16-dUTP) was incorporated into mitochondrial rDNA and small nuclear U1 probes by nick-translation. The probes were hybridized to sections of Drosophila ovaries and subsequently detected with an anti-biotin antibody and protein A-gold complex. On semi-thin sections, probe detection was achieved by amplification steps with anti-protein A antibody and protein A-gold with subsequent silver enhancement. At the electron microscope level, specific labeling was obtained over structures known to be the site of expression of the appropriate genes (i.e., either over mitochondria or over nuclei). The labeling pattern at the light microscope level (semi-thin sections) was consistent with that obtained at the electron microscope level. The described nonradioactive procedures for hybrid detection on Lowicryl K4M-embedded tissue sections offer several advantages: rapid signal detection: superior morphological preservation and spatial resolution; and signal-to-noise ratios equivalent to radiolabeling.     

Identification of functional murine adenosine deaminase cDNA clones by complementation in Escherichia coli

Total poly(A+) RNA derived from a mouse cell line with amplified adenosine deaminase genes was used as template to synthesize double-stranded cDNA. The cDNAs were inserted into the PstI site of the beta-lactamase gene in plasmid pBR322 following G-C tailing. After transformation into adenosine deaminase-deficient Escherichia coli hosts, recombinant plasmids containing functional murine adenosine deaminase cDNAs were identified by selecting for functional complementation. Analysis of plasmids containing functional adenosine deaminase cDNA sequences strongly suggested that adenosine deaminase expression resulted mainly from beta-lactamase/adenosine deaminase fusion proteins even when the adenosine deaminase codons were out-of-frame with respect to the beta-lactamase gene codons upstream. The nucleotide sequence of a 1.65-kilobase pair cDNA insert in one of the functional recombinant clones was determined and found to contain a 1056-nucleotide open reading frame. When this 1056-nucleotide open reading frame was inserted into a mammalian expression vector and introduced into monkey kidney cells, a high level of authentic mouse adenosine deaminase was produced. Nucleic acid blot analysis using a full-length adenosine deaminase cDNA clone as probe revealed that the mouse adenosine deaminase structural gene was at least 21 kilobase pairs in size and encoded three polyadenylated mRNAs. Analysis of the cDNA library from which the functional clones were isolated suggested that this approach of cloning functional mammalian adenosine deaminase cDNA clones by genetic complementation of enzyme-deficient bacteria could be accomplished even if the abundance of the adenosine deaminase mRNA sequences were as low as approximately 0.001%.     

Covalent linkage of 125I-insulin to a cytosolic insulin-degrading enzyme

Cytosol extracts high in insulin-degrading activity were cross-linked to 125I-insulin with the bifunctional cross-linker disuccinimidyl suberate. With cytosols from either rat muscle, liver, kidney or brain or human erythrocytes, only a single protein (Mr = 110,000) was specifically labeled. Three different lines of evidence indicated that this labeled protein is insulin-degrading enzyme, a cysteine protease which accounts for most of the insulin-degrading activity in cell extracts. Firstly, the cross-linking of 125I-insulin to this protein is inhibited by unlabeled insulin over the same concentration range of insulin which inhibits degradation. Separated insulin A and B chain were less potent at inhibiting cross-linking, whereas bovine serum albumin and cytochrome c were without effect. Secondly, antibodies to purified insulin-degrading enzyme precipitated the labeled protein in parallel with their ability to precipitate the insulin-degrading activity of the extracts. Thirdly, when the insulin-degrading activity was purified 40,000-fold from erythrocytes, this Mr 110,000 protein co-purified. These results indicate that cross-linking 125I-insulin may be a convenient method for labeling the insulin-degrading enzyme.     

Photoaffinity labeling of a viral induced protein from tobacco. Characterization of nucleotide-binding properties

We have used the photoaffinity analogs 8-azidoadenosine 5'-triphosphate (8-N3ATP) and 8-azidoguanosine 5'-triphosphate (8-N3GTP) to investigate the relationship between a viral induced protein (Mr = 120,000) in tobacco mosaic virus (TMV)-infected tobacco and the TMV-induced RNA-dependent RNA polymerase activity. When the radioactive analogs [gamma-32P]8-N3ATP and [gamma-32P]8-N3GTP were incubated with the tobacco tissue homogenate from TMV-infected plants, incorporation of label occurred into the viral induced protein in the presence of UV light. The incorporation was found to be totally dependent on UV-illumination and greatly enhanced by Mg2+. Saturation of photoincorporated label indicates an apparent Kd of 16 microM (+/- 3 microM) and 12 microM (+/- 3 microM) for 8-N3ATP and 8-N3GTP, respectively. Protection against photolabeling by [gamma-32P]8-N3ATP and [gamma-32P]8-N3GTP with various nonradioactive nucleotides and nucleosides suggests that the photolabeled site is protected best by nucleoside triphosphates. At 200 microM both deoxyribonucleoside triphosphates and ribonucleoside triphosphates were very effective at protecting the site from photolabeling. These data suggest that the photolabeled protein may be part of an RNA-dependent RNA polymerase. The utility of nucleotide photoaffinity analogs as a method to study viral induced nucleotide-binding proteins is discussed.