Complement-dependent lysis of tumor cells by a baboon IgM antibody to a tumor-associated antigen

Summary We developed a high-titer polyclonal antiserum to a glycoprotein tumor-associated antigen (TAA) by immunization of a baboon with the purified glycoprotein antigen. The baboon serum was fractionated into IgG and IgM components by DEAE Affi-Gel blue chromatography. The ability of the baboon IgM anti-TAA antibody to effect tumor cell lysis in the presence of complement was tested using a chromium-release assay. The baboon antibody was able to lyse melanoma target cells (20.8%–71.4% cytolysis), breast carcinoma cells (36.5%–38.9% cytolysis), and a neuroblastoma cell line (35.5% cytolysis) in the presence of complement but did not effect significant lysis of autologous lymphoblastoid cell lines (4.9% cytolysis) or peripheral blood lymphocytes from healthy volunteers (12.6% cytolysis). Cytolysis of melanoma target cells was completely inhibited by preabsorption of the IgM anti-TAA antibody with UCLA-SO-M14 (M14) cells and partially inhibited by preabsorption with several other melanoma cell lines. There was no significant inhibition of tumor cell lysis after preabsorption of the antibody with lymphoblastoid cell lines. Complement-dependent lysis of M14 targets could be blocked by addition of the purified antigen to the antibody prior to incubation with the tumor cells. Our results suggest that the glycoprotein TAA resides on the tumor cell surface and that the baboon IgM anti-TAA antibody recognizes the antigen on the cell surface and is able to fix complement and effect the lysis of the tumor cells.     

Crystal structure of the Y52F/Y73F double mutant of phospholipase A2: increased hydrophobic interactions of the phenyl groups compensate for the disrupted hydrogen bonds of the tyrosines.

The enzyme phospholipase A2 (PLA2) catalyzes the hydrolysis of the sn-2 ester bond of membrane phospholipids. The highly conserved Tyr residues 52 and 73 in the enzyme form hydrogen bonds to the carboxylate group of the catalytic Asp-99. These hydrogen bonds were initially regarded as essential for the interfacial recognition and the stability of the overall catalytic network. The elimination of the hydrogen bonds involving the phenolic hydroxyl groups of the Tyr-52 and -73 by changing them to Phe lowered the stability but did not significantly affect the catalytic activity of the enzyme. The X-ray crystal structure of the double mutant Y52F/Y73F has been determined at 1.93 A resolution to study the effect of the mutation on the structure. The crystals are trigonal, space group P3(1)21, with cell parameters a = b = 46.3 A and c = 102.95 A. Intensity data were collected on a Siemens area detector, 8,024 reflections were unique with an R(sym) of 4.5% out of a total of 27,203. The structure was refined using all the unique reflections by XPLOR to a final R-factor of 18.6% for 955 protein atoms, 91 water molecules, and 1 calcium ion. The root mean square deviation for the alpha-carbon atoms between the double mutant and wild type was 0.56 A. The crystal structure revealed that four hydrogen bonds were lost in the catalytic network; three involving the tyrosines and one involving Pro-68. However, the hydrogen bonds of the catalytic triad, His-48, Asp-99, and the catalytic water, are retained. There is no additional solvent molecule at the active site to replace the missing hydroxyl groups; instead, the replacement of the phenolic OH groups by H atoms draws the Phe residues closer to the neighboring residues compared to wild type; Phe-52 moves toward His-48 and Asp-99 of the catalytic diad, and Phe-73 moves toward Met-8, both by about 0.5 A. The closing of the voids left by the OH groups increases the hydrophobic interactions compensating for the lost hydrogen bonds. The conservation of the triad hydrogen bonds and the stabilization of the active site by the increased hydrophobic interactions could explain why the double mutant has activity similar to wild type. The results indicate that the aspartyl carboxylate group of the catalytic triad can function alone without additional support from the hydrogen bonds of the two Tyr residues.     

Toxicity and moniliformin production by four recently described species of Fusarium and two uncertain taxa

Four recently described species, Fusarium nygamai, F. dlamini, F. beomiforme and F. napiforme and two uncertain taxa, F. nygamai from millet in Africa and Fusarium species from rice with Bakanae disease, were tested for toxicity and moniliformin production. Cultures grown on autoclaved corn were fed to groups of four one-day-old ducklings for 14 days. Isolates that caused the death of 3 or 4 out of 4 ducklings were considered to be toxic and analyzed for moniliformin. All 15 isolates of F. dlamini tested were nontoxic. The other taxa contained some isolates that were toxic to ducklings and produced moniliformin in corn cultures. This is the first report of moniliformin production by F. beomiforme (200–890 g/g), and F. napiforme (16–388 g/g), and by F. nygamai not obtained from millet in Africa (15–874 g/g). The highest production of moniliformin was obtained from the 19 isolates of F. nygamai from millet in Africa (4300–18200g/g) and the 15 isolates from rice with Bakanae disease (2300–19300 g/g). The taxonomic position of these two uncertain taxa should be re-evaluated.     

Urea metabolism in the cyanobacterium Anabaena cycadeae: regulation of urea uptake and urease by ammonia

A total of 160 Escherichia coli positive for F165 fimbrial antigen and isolated from diarrheic and septicemic animals, were examined for the presence of the pap, afa, and sfa/foc operons or related nucleotide sequences using colony hybridization. Most isolates shared DNA sequences with the pap operon sequences alone or in association with afa or sfa. Thus, our results indicate that F165-positive E. coli from diseased animals share DNA sequences with operons coding for adhesins important in human extra-intestinal disease and that multiple adhesin systems are often found in single isolates. However, 20% of the F165-positive isolates did not show any homology with the probes representing the three adhesin systems, suggesting that one of the operons responsible for F165 production could be different from the pap, sfa/foc, and afa operons.     

31P NMR analysis of intracellular pH of Swiss mouse 3T3 cells: Effects of extracellular Na+ and K+ and mitogenic stimulation

Summary Swiss mouse 3T3 cells grown on microcarrier beads were superfused with electrolyte solution during continuous NMR analysis. Conventional³¹P and¹⁹F probes of intracellular pH (pH _c ) were found to be impracticable. Cells were therefore superfused with 1 to 4mm 2-deoxyglucose, producing a large intracellular, pH-sensitive signal of 2-deoxyglucose phosphate (2DGP). The intracellular incorporation of 2DGP inhibited the Embden-Meyerhof pathway. However, intracellular ATP was at least in part retained and the cellular responsivity to changes in extracellular ionic composition and to the application of growth factors proved intact. Transient replacement of external Na⁺ with choline or K⁺ reversibly acidified the intracellular fluids. Quiescent cells and mitogenically stimulated cells displayed the same dependence of shifts in pH _c on external Na⁺ concentration (c _Na ^o ). pH _c also depended on intracellular Na⁺ concentration (c _Na ^o ). Increasingc _Na ^c by withdrawing external K⁺ (thereby inhibiting the Na,K-pump) caused reversible intracellular acidification; subsequently reducingc _Na ^o produced a larger acid shift in pH _c than with external K⁺ present. Comparison of separate preparations indicated that pH _c was higher in stimulated than in quiescent cells. Transient administration of mitogens also reversibly alkalinized quiescent cells studied continuously. This study documents the feasibility of monitoring pH _c of Swiss mouse 3T3 cells using³¹P NMR analysis of 2DGP. The results support the concept of a Na/H antiport operative in these cells, both in quiescence and after mitogenic stimulation. The data document by an independent technique that cytoplasmic alkalinization is an early event in mitogenesis, and that full activity of the Embden-Meyerhof pathway is not required for the expression of this event.     

Association of Criconemella xenoplax and Fusarium spp. with Root Necrosis and Growth of Peach

Criconemella xenoplax, Fusarium solani, and F. oxysporum caused necrosis of Nemaguard peach feeder roots in greenhouse tests. Root necrosis was more extensive in the presence of either fungus than wtih C. xenoplax alone. Shoot growth and plant height were less for plants inoculated with F. oxysporum or F. solani than for plants inoculated with the fungi plus C. xenoplax. Neither synergistic nor additive effects on root necrosis or plant growth occurred between C. xenoplax and the fungal pathogens.     

Effects of Hypoxia and Anoxia on the Ex Vivo Release of Prostaglandins from Mouse Cortical Slices

Arachidonic acid is transiently accumulated in the brain as a result of a variety of pathological conditions. The synthesis and release of some of its metabolites, namely, prostaglandin E2 (PGE2), thromboxane B2 (TXB2), and 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) from cortical slices of mice were studied following exposure to 6 min of hypoxia (7% O2), 45 s of anoxia, and 5 min-4 h of reoxygenation following anoxia. Hypoxia induced a slight increase in the rate of TXB2 release and a slight decrease in the rate of PGE2 release, whereas 6-keto-PGF1 alpha was unaffected. Anoxia (45 s) followed by reoxygenation induced a transient increase in the release of PGE2 and of 6-keto-PGF1 alpha with a return to the normal rate at 30 min and 2 h of recovery, respectively. However, the rate of TXB2 synthesis and release reached its peak (twofold increase) after 1 h and remained significantly higher than the control rate even after 4 h of normal air breathing. Our results demonstrate that hypoxia and anoxia, even of short duration, selectively trigger the activity of thromboxane synthetase and that this elevated rate of synthesis and release persists long after normal oxygen supply is restored. We suggest that enhanced thromboxane synthesis, with normal prostacyclin levels, might have a role in the pathophysiology of ischemic cell damage.     

Major Central Nervous System Myelin Glycoprotein of the African Lungfish (Protopterus dolloi) Cross-Reacts with Myelin Proteolipid Protein Antibodies, Indicating a Close Phylogenetic Relationship with Amphibians

CNS myelin was isolated from the spinal cord of the African lungfish Protopterus dolloi. Its proteins consisted of (1) two basic proteins (16,000 and 18,500 apparent Mr) that reacted with anti-human CNS myelin basic protein antibodies and (2) a major protein (29,000 apparent Mr) that stained with concanavalin A-horseradish peroxidase and bound to anti-rat CNS myelin proteolipid protein (PLP) antibodies. This dominant 29,000 Mr protein showed no reaction with antibodies against the major bovine PNS myelin glycoprotein P0. Following treatment with endoglycosidase F the 29,000 Mr protein was reduced in size to a 26,000 apparent Mr component that no longer bound concanavalin A but retained the anti-PLP reactivity. These results agree with a concanavalin A-binding oligosaccharide linked through asparagine to a protein backbone of PLP homology. The major 29,000 Mr lungfish CNS myelin protein was therefore termed g-PLP (glycosylated proteolipid protein). This is the first report demonstrating the occurrence of a PLP-cross-reactive protein in CNS myelin of a fish. It attests to the close phylogenetic relationship of lungfishes to amphibians. Amphibians were previously recognized as the oldest class bearing PLP in its CNS myelin.     

Prenylated bianthrones and vismione F from Psorospermum febrifugum

The roots of Psorospermum febrifugum collected in Malawi contained together with the known vismione D and geranyloxyemodin four new compounds: vismione F and the three bianthrones A₁, A_3a and A_3b. All the isolated compounds contained C- or O-geranyl substituents and showed a close biogenetic relationship.     

Trypanosoma brucei: analysis of relapsing populations in sensitive and resistant breeds of cattle

The clone DiTat 1.1 of Trypanosoma brucei brucei was injected into four bovids, and clones obtained from successive waves of parasitemia were used to study the expressed variant-specific surface glycoprotein repertoire. Twenty-four clones were obtained which could be classified into 12 different variable antigen types, in addition to the clone injected, using agglutination or immunofluorescence with monospecific antisera. The variable surface glycoproteins of the 25 clones were extracted using the detergent octyl-beta-D-glucopyranoside in the presence of the protease inhibitor, N-cbz-L-phenylalaninechloromethylketone. The molecular weights varied from 52,000 to 69,000 and the pI from 5.0 to 8.8. The virulence of 14 clones representing 13 variable antigen types was ascertained in mice. The mean survival time ranged from 20.5 to 43.0 days. Clones isolated from early peaks of parasitemia in the bovid were the most virulent while clones derived from later peaks were less virulent. It seems that organisms of diminishing virulence appear in bovids, leading to self-cure of the disease. All clones were sensitive to human serum in a blood infectivity inhibition test. Antibody against all virulent clones appeared in 20 cattle (10 Zebus, 10 Baoulés) which had been injected with T. brucei DiTat 1.1. There was no evidence for parasites of high or low virulence being preferentially expressed in resistant or sensitive hosts.