Studies on the mechanism of natural killer cell-mediated cytotoxicity. VII. functional comparison of human natural killer cytotoxic factors with recombinant lymphotoxin and tumor necrosis factor

The present study was undertaken to evaluate the possible contribution of other cytokines to the lytic activity of NKCF-containing supernatants. We compared some of the functional properties of human NKCF and purified recombinant human rLT and rTNF. It was found that the target cell specificity of rLT was quite different from NKCF in that rLT was neither species specific nor NK specific. Furthermore, antibodies against rLT did not affect the lytic activity of NKCF. These results demonstrate that LT does not significantly contribute to the lytic activity mediated by NKCF. The target specificity of rTNF was found to be related to that of NKCF with the exception of one NK-resistant cell line that was lysed by rTNF in a 20-hr 51Cr-release assay. However, rTNF was not toxic to any of the target cells tested as assessed by trypan blue exclusion in a 20-hr assay unless the targets were labeled with 51Cr. In contrast, NKCF did kill target cells as detected by trypan blue exclusion that were not labeled with 51Cr. Further analysis of this mechanistic difference in the lytic activity of rTNF and NKCF revealed that rTNF in combination with either cycloheximide or mitomycin C but not IFN-gamma could lyse unlabeled U937 target cells. In addition, pretreatment of U937 target cells with nonradioactive Na2CrO4 at concentrations equivalent to that used to 51Cr-labeled cells resulted in their susceptibility to lysis by rTNF as assessed by trypan blue exclusion. These findings suggest that lysis of several susceptible target cells in 20 hr by rTNF requires the presence of additional agents that may be sublethally toxic and/or inhibitory to macromolecular synthesis. Antibody inhibition studies revealed that anti-TNF mediated from partial to complete inhibition of lysis of U937 by unfractionated supernatants containing NKCF. However, fractionation of such supernatants on chromatofocusing columns yielded two distinct peaks of activity eluting in the pH range of 5 to 6 and 7 to 8. Anti-TNF could inhibit the acidic form of NKCF but not the neutral form. It is concluded that NKCF activity is mediated in part by TNF or an antigenically related molecule as well as some other distinct factor(s). The lack of consistent inhibition of NK CMC by anti-TNF suggests that TNF alone is not sufficient to mediate NK activity, or else it is inaccessible to the added antibody.     

Assignment of resonances in the 1H nuclear magnetic resonance spectrum of the carbon monoxide complex of sperm whale myoglobin by phase-sensitive two-dimensional techniques

Phase-sensitive two-dimensional nuclear magnetic resonance (n.m.r.) experiments have been used to obtain extensive proton resonance assignments for the carbon monoxide complex of sperm whale myoglobin. Multiple quantum experiments were particularly important in the assignment procedure. The assignments are the most complete yet reported for a protein of such high molecular weight (approximately 18,000) and make possible new and comprehensive studies of the structure and dynamics of carbonmonoxymyoglobin in solution. Assignments for seven of the histidine residues are reported, including the critical proximal and distal histidines. Most of these are at variance with the assignments already in the literature. The present n.m.r. data indicate that histidines 24 (B5) and 119 (GH1) are hydrogen bonded to each other and, in contrast to neutron diffraction data, show that His24 does not protonate at pH greater than 5. The aromatic rings of all the phenylalanine and tyrosine residues undergo rapid flips about the ring axis. The side-chains of Leu89 (F4) and Phe138 (H15), which border a large hydrophobic cavity, are particularly mobile.     

l-Alanine uptake in brush border membrane vesicles from the gill of a marine bivalve

Summary Brush border membrane vesicles were prepared from mussel gills using differential and sucrose density gradient centrifugation. These vesicles contained both the maximal Na⁺-dependent alanine transport activity found in the gradient and the maximal activities of -glutamyl transpeptidase and alkaline phosphatase. Electron micrographs showed closed vesicles of approximately 0.1–0.5 m diameter. Transport experiments using these vesicles demonstrated a transient 18-fold overshoot in intravesicular alanine concentration in the presence of an inwardly directed Na⁺ gradient, but not under Na⁺ equilibrium conditions. A reduced overshoot (10-fold) was seen with an inwardly directed K⁺ gradient. Further studies revealed a broad cation selectivity, with preference for Na⁺, which was characteristic of alanine transport but not glucose transport in these membranes. The apparent amino acid specificity of the uptake pathway(s) was similar to that of intact gills and supported the idea of at least four separate pathways for amino acid transport in mussel gill brush border membranes. The apparent Michaelis constant for alanine uptake was approximately 7m, consistent with values forK _t determined with intact tissue.     

Refinement of the crystal structure of wheat germ agglutinin isolectin 2 at 1.8 A resolution

The crystal structure of wheat germ agglutinin isolectin 2 has been refined by the restrained least-squares method of Hendrickson & Konnert (1980). The asymmetric unit of the C2 crystals contains two chemically identical promoters related by a non-crystallographic 2-fold screw operation. A total of 2290 protein atoms and 186 ordered water sites refined to a final R-factor of 0.179 and an average B-value of 21.6 A2, using 54% (15,601) of the total possible number of reflections in the resolution range 8 to 1.8 A with Fo greater than 3 sigma (Fo). The final model conforms to stereochemically correct bond distances and angles with root-mean-square (r.m.s.) values of 0.018 A and 3.3 degrees, respectively. Accuracy of this model is estimated to be 0.20 A on the basis of a Luzzati plot. Main-chain atomic positions in the two independent promoters, designated I and II, agree with an r.m.s. deviation of 0.30 A (0.58 A for all atoms), indicating identical backbone conformation. The largest discrepancies are seen at flexible surface residues. One error was detected in the amino acid sequence at position 41 (Ser), which refined satisfactorily as a Trp. Loss of electron density for residue A171 during the course of refinement suggests either disorder or absence of this C-terminal residue. The conformation of the polypeptide chain, which is folded into four homologous 43-residue domains (A, B, C and D), was analyzed in terms of dihedral angles, backbone hydrogen bond lengths and CA-atom positions. The four domains were found to be very similar according to all these criteria and superposition of their CA-atoms yielded r.m.s. distances ranging from 0.36 to 0.72 A for the six possible comparisons [corrected]. Large deviations (greater than 1.0 A) are only seen in the five-residue segments that link adjacent domains and at the N and C termini. Refinement has also allowed critical examination of each of the two unique sugar binding sites, referred to as "primary" and "secondary" sites, in different lattice environments. While the essential tyrosyl side-chain in each of these sites (Y73, Y159) assumes precise orientation for optimum hydrophobic contact with the N-acetyl methyl group of the sugar ligand, side-chains involved in hydrogen bonds (S62, E115; and S148, D29) were found to be relatively flexible and able to adapt their conformation to changes in environment. Ordered water structure present in these binding sites is not completely analogous in the different environments.(ABSTRACT TRUNCATED AT 400 WORDS)     

Resolution of pea legumin subunits by high-performance liquid chromatography

Pea legumin was dissociated into its component subunits by 6 M urea: these were subsequently fractionated by FPLC using a combination of Mono P, Mono Q, and Mono S columns. The resolution and speed of separation were greatly improved in comparison with previous fractionations. Twelve discrete fractions were obtained and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Six "normal" legumin subunits (Mr 60,000) were identified as well as some "large" (Mr 66,000) and "small" (Mr 44,000) subunits. A few polypeptides of unknown origin were also observed. Four subunits were purified to homogeneity as adjudged by electrophoresis and HPLC and in sufficient yields to permit further studies. Anomalous electrophoretic behavior of the legumin subunits was also observed.     

Examination of the Na+-induced conformational change of the intestinal brush border sodium/glucose symporter using fluorescent probes

The Na+-induced change in conformation of the intestinal brush border glucose carrier has been examined by three procedures. In the first, we have measured the effect of Na+ on the binding of fluorescein isothiocyanate (FITC) to the glucose site; 100 mM Na increased the specific [blocked by D-glucose, p-(chloromercuri)benzenesulfonic acid, and N-acetylimidazole] FITC binding to a 75-kilodalton polypeptide 3-fold. In the second series, we have examined the effect of Na+ on the susceptibility of the fluorescently labeled glucose site [pyrene isothiocyanate (PYTC) labeled] to a hydrophilic quencher (Tl+); 100 mM NaCl increased the fraction of PYTC sites available to Tl+ from 32% to 92% and decreased the apparent quenching constant from 94 to 44 M-1. Finally, in the third series, we probed the distribution of tryptophan residues 15-30 A from the glucose site using a "distant reporter group method", where tryptophan was used an an energy donor to anthracene isothiocyanate bound to the glucose site. Tryptophan quench reagents (I-, Cs+, and acrylamide) were then employed to probe the accessibility of the glucose site tryptophans in the presence and absence of sodium. In the absence of Na+, there were two major classes of glucose tryptophans--exterior surface residues and residues buried in the hydrophobic protein matrix. Na+ caused a redistribution of the donor tryptophans such that a higher percentage were accessible to I- (51% vs. 25%) and fewer were accessible to Cs+ (13% vs. 25%) and acrylamide (27% vs. 57%).(ABSTRACT TRUNCATED AT 250 WORDS)     

Evolution of the 28S ribosomal RNA gene in anurans: regions of variability and their phylogenetic implications

Fifteen restriction sites were mapped to the 28S ribosomal RNA gene of individuals representing 54 species of frogs, two species of salamanders, a caecilian, and a lungfish. Eight of these sites were present in all species examined, and two were found in all but one species. Alignment of these conserved restriction sites revealed, among anuran 28S rRNA genes, five regions of major length variation that correspond to four of 12 previously identified divergent domains of this gene. One of the divergent domains (DD8) consists of two regions of length variation separated by a short segment that is conserved at least throughout tetrapods. Most of the insertions, deletions, and restriction-site variations identified in the 28S gene will require sequence-level analysis for a detailed reconstruction of their history. However, an insertion in DD9 that is coextensive with frogs in the suborder Neobatrachia, a BstEII site that is limited to representatives of two leptodactylid subfamilies, and a deletion in DD10 that is found only in three ranoid genera are probably synapomorphies.      

Stem cell maintenance and commitment to differentiation in long-term cultures of murine marrow obtained from different spatial locations in the femur

Abstract. Murine bone marrow was separated into axial and marginal fractions in order to investigate the ability of cells from different spatial locations in the marrow to establish long-term cultures. The maintenance of haemopoiesis was significantly poor in long-term cultures of marginal marrow compared with axial or control (unfractionated marrow) cultures. Using techniques to further fractionate the axial or marginal marrow by depleting either stromal or haemopoietic cells, it was possible to investigate the relative importance of stromal and haemopoietic cell components. In the combinations studied, the more important determinant of effective in vitro haemopoiesis was the source of the haemopoietic cells rather than the stroma. The most effective stem cell maintenance and commitment to differentiation was observed when the source of the haemopoietic population was axial marrow. The data are consistent with axial marrow being a source of 'high quality' stem cells and this quality being an intrinsic property of the cells rather than one imposed by the stromal environment.     

Variability in Effectiveness of Rhizobia during Culture and in Nodules

The ability of three strains of Bradyrhizobium sp. (Vigna) to fix dinitrogen in symbiotic association with siratro (Macropitilium atropurpureum) was measured after culture in broth and after isolation from nodules. Seven transfers were made between the initial broth culture and the final broth culture. A total of 40 single-colony isolates were obtained from cultures 1 and 7 to test effectiveness. Variation in dinitrogen-fixing effectiveness of the population of one strain did not change on culturing, whereas there was considerable variation in effectiveness of populations of the other two strains. Generally, single-colony isolates from individual nodules had similar levels of effectiveness, but some exceptions occurred. Isolates from different nodules formed by the same Bradyrhizobium strain often differed in their effectiveness.     

Nonoxidative ethanol metabolism in human leukocytes: detection of fatty acid ethyl ester synthase activity

Oxidative pathways of alcohol metabolism such as alcohol dehydrogenase usually are not present in human blood and therefore clinical studies correlating ethanol metabolism with alcohol abuse syndromes have not been performed. To assess the activity of nonoxidative ethanol metabolism in blood, we assayed for the activity of fatty acid ethyl ester synthase, a pathway recently described as abundant in the human organs most commonly damaged by alcohol. Indeed, peripheral human leukocytes contain detectable fatty acid ethyl ester synthase activity: 1.2 X 10(6) leukocytes from 10 ml blood catalyze the synthesis of ethyl oleate at 1.4 nmol/4 hr. The reaction is linear with respect to cell number and expended time; Km oleate = 600 microM, Km ethanol = 600 mM. DEAE cellulose chromatography partially purifies synthase activity into a minor and major form (activity ratio = 10/1). Thus, gene products exist in human blood that recognize ethanol and whose biological activity is conveniently assayable for clinical investigations of alcohol metabolism and abuse.