Bioassay of a β-exotoxin of Bacillus thuringiensis against Heliothis zea larvae

A β-exotoxin of Bacillus thuringiensis was bioassayed on 1st-, 3rd-, and 4th-instar Heliothis zea larvae. Larvae were fed continuously on diet incorporated with concentrations of 1–700 μg AI/ml diet. Larval and/or pupal death was the measured response criterion. Dosage-mortality responses were determined at two evaluation times, 7 days post-initiation and after the entire larval-pupal development period, using probit analysis procedures. The LC₅₀ values for 1st-, 3rd-, and 4th-instar larvae at these two evaluation times were 4.9, 134.6, and 286.2 μg AI/ml diet, and 4.0, 17.6, and 66.4 μg AI/ml diet, respectively. Differences in responses between instars were more pronounced at 7 days than after the entire development period. The LT₅₀ values for 1st-, 3rd-, and 4th-instar larvae decreased from 7.1 to 3.1, 12.7 to 5.4, and 11.6 to 5.2 days post-initiation, respectively, as dosages were increased. The toxin did not act as a feeding deterrent, as all increases in dosage caused increases in mortality. Nineteen and 38% of those 3rd- and 4th-instar larvae, respectively, which survived β-exotoxin intoxication pupated later than untreated cohorts.     

Iron transport in Salmonella typhimurium: mutants blocked in the biosynthesis of enterobactin

A number of mutants of Salmonella typhimurium were isolated which are blocked in the biosynthesis of enterobactin, an iron chelator that is secreted by the wild-type bacteria when they are grown on low iron media. One class of these enb mutants accumulates the enterobactin precursor 2,3-dihydroxybenzoic acid, and another class does not accumulate any detectable catechol precursor. The enb mutants grow very well on a glucose-mineral salts medium. Addition of citrate, itself an iron chelator, to the medium drastically inhibits growth unless the medium is supplemented with enterobactin or iron salts. Citrate inhibits iron uptake from the medium by enb mutants; enterobactin counteracts this inhibition and also, by itself, increases iron uptake. Thus, the apparent function of enterobactin is to promote the absorption of iron from the medium by the bacteria. Transduction experiments showed that the genes for enterobactin biosynthesis are closely linked on the S. typhimurium chromosome. It is suggested that they form an operon which is repressed by the presence of iron. S. typhimurium can utilize the iron chelate ferrichrome. (Deferriferrichrome is a cyclic hexapeptide that is produced by some fungi but not by S. typhimurium.) The enb mutants use ferrichrome as an effective growth factor.     

Enriched Selection of Dominant Mutations: Histidine Operator Mutations

In the course of selection of bacteria with derepressed levels of histidine biosynthetic enzymes, it was found that when mutagen-treated cells were spread on a selective medium without allowing intervening growth to occur, the frequency of operator mutants obtained was dramatically increased. This may be useful as a general enrichment for operator or other dominant mutations.     

Active specific immunotherapy of Dukes B2 and C colorectal carcinoma: Comparison of two doses of the vaccine

Summary The ability of active specific immunotherapy to enhance immune responses to autologous tumor-associated antigens (TAA) and to prolong the disease-free interval was evaluated in patients with Dukes B₂ and C colorectal carcinoma who had undergone potentially curative resections. Patients were sensitized in the early postoperative period with irradiated autologous adenocarcinoma cells mixed with bacillus Calmette-Guérin (BCG) to yield either a low-dose vaccine (3×10⁶ tumor cells) or a high-dose vaccine (1×10⁷ tumor cells). Six of seven patients who received the low-dose vaccine developed delayed-type hypersensitivity (DTH) responses to autologous tumor cells upon completion of the vaccination, whereas all four patients receiving high-dose vaccine displayed a positive DTH response. However, DTH responses to autologous TAA waned within 3 months in all patients receiving the low-dose vaccine; DTH responses persisted for 3 months in three of the four high-dose vaccine patients. In vitro lymphoproliferative responses to TAA correlated with DTH responses to autologous tumor cells. Active specific immunotherapy appeared to induce specific immune responses either in vitro or in vivo to autologous TAA because it did not induce responses to autologous mucosa cells. There were no complications caused by BCG or tumor cells. This series demonstrates that active specific immunotherapy is a nontoxic treatment that augments immunity to autologous TAA.This project was supported by grants from Cutter Laboratories, Inc., the Annual Campaign of the University of Texas System Cancer Center, and the National Cancer Cytology Center     

Immunoregulatory factors derived from human tumors. II. Partial purification and further immunobiochemical characterization of a human sarcoma-derived immunosuppressive factor expressing HLA-DR and immunoglobulin-related determinants

An immunoregulatory factor (IRF) that suppresses Con A-mediated peripheral blood mononuclear cell (PBM) proliferative responses was partially purified by DEAE anion exchange chromatography and affinity chromatography from a 3 M KCl extract of a human liposarcoma. The factor (m.w. = 70K) co-purified with albumin, monitored by two-dimensional gel electrophoresis, and demonstrated a heterogeneous isoelectric point (pI 7.6-7.8). Xenoantisera produced against the DEAE-purified fraction and coupled to Affigel 10 removed suppressive activity that could subsequently be eluted by glycine-HCl, pH 3.5. An anti-albumin column partially removed activity, but the unbound 70K factor could still be detected in the column effluent. A xenoantiserum to this 70K effluent coupled to acrylamide beads completely removed the immunosuppressive activity in immunodepletion experiments. Further direct binding enzyme-linked immunoassays (ELISA) and solid-phase immunoabsorption experiments with monoclonal antibodies to human anti-HLA-DR framework determinants and a constant region of the IgM mu-chain demonstrated determinants on the 70K factor recognized by these antibodies.     

A quantitative morphological study of osmotically induced swelling and shrinkage in nervous tissue

The rabbit retina was used to study, in vitro, the responses of central nervous tissue to changes in extracellular osmolarity. After isolation, retinas were incubated in either hypertonic or hypotonic medium containing 80 milliosmols more or 80 milliosmols less sodium chloride than the isotonic control medium. After fixation and embedding, comparable areas of each retina were sectioned and studied with the phase and electron microscopes. The diameters of receptor cell inner segments, synapses, nuclei, and mitochondria were measured on micrographs; mean nuclear areas and volumes were calculated. Cutouts from micrographs also provided areas and volumes of the receptor cell nucleus and its 'surround' of axons, dendrites, glial processes, and extracellular space. In general, hypertonic incubation produced decreases in the linear dimensions, areas, and volumes of the receptor cell, its nucleus, and its mitochondria that were consistent with their behaviour as osmometers. After hypotonic incubation, the increases in the diameters of inner segments, synapses, and mitochondria were in the predicted range. The increases for the nuclei themselves, and the nuclei and their 'surround' were less than expected. This may have been due to the failure of the preparative techniques to maintain the swollen state of these larger structures.     

Glutaraldehyde fixation of central nervous tissue: an electron microscopic evaluation in the isolated rabbit retina

The isolated rabbit retina was studied electron microscopically after fixation with a 3% solution of glutaraldehyde in a 0.05 M S?rensen's phosphate buffer. In radial sections, the inner segments, nuclei, and synapses of the photoreceptor cells seemed similar in size to those from retinas that had been fixed in an isotonic solution containing 1 % crystalline osmium tetroxide in the incubating medium used for the isolation procedure. However, when the number of comparable structures was greatly increased by viewing them in tangential sections, the cellular shrinkage and mitochondrial swelling produced by this widely used, hypertonic, glutaraldehyde fixative were obvious.