Immune reactivity in cattle with ocular squamous cell carcinoma after intralesional BCG immunotherapy

Summary Lymphocyte stimulation with Con A and specific immune reactivity to BCG (antibody formation to BCG and DTH reaction to PPD) were determined in BCG-treated, surgically treated and untreated cows with ocular squamous cell carcinoma. In tumor-bearing cows the Con A-induced proliferation of lymphocytes was reduced when compared to healthy controls. This suppression consisted of a reduced blastogenic response to Con A of lymphocytes from tumor-bearing cows, and the presence of a factor in the sera of these animals, as these sera suppressed the blastogenic response of lymphocytes from healthy cows. BCG had only a minor influence on the suppressive activity. Antibodies to BCG were demonstrated in 50% of the BCG-treated animals. The formation of antibodies was not influenced by intradermal injection of PPD of Mycobacterium bovis. Absorption of a BCG antibody containing serum with BOSCC tumor extracts did not reveal the existence of cross reacting antigens between BCG and BOSCC. Pretherapeutic and posttherapeutic Con A reactivity could not be correlated with clinical response. Of the 30 BCG treated cows 29 developed a positive DTH reaction to PPD. Correlation between clinical response and immune reactivity was seen only with regard to the DTH reaction to PPD: this reaction remained positive for a longer period after treatment in animals with a favorable clinical outcome than in nonresponding animals.Animals were maintained under the guidelines laid down by the Faculty of Veterinary Medicine, State University, Utrecht, The NetherlandsGrant recipient of the Koningin Wilhelmina Fonds (Netherlands Cancer Foundation) Abbreviations used: BCG, Bacillus Calmette-Guerin; BOSCC, bovine ocular squamous cell carcinoma PBL peripheral blood leukocytes; PPD, purified protein derivative of Mycobacteria; DTH, delayed type hypersensitivity Con A, concanavalin A; PHA, phytohemagglutinin; PWM, pokeweed mitogen     

Bacteriological survey of sixty health foods.

A bacteriological survey was performed on 1,960 food samples encompassing 60 types of health foods available in the Baltimore-Washington, D.C., metropolitan area. No consistent bacteriological distinction (aerobic plate counts, total coliform and fecal coliform most probable numbers) was observed between foods labeled as organic (raised on soil with compost or nonchemical fertilizer and without application of pesticides, fungicides, and herbicides) and their counterpart food types bearing no such label. Types and numbers of samples containing Salmonella were: sunflower seeds, 4; soy flour, 3; soy protein powder, 2; soy milk powder, 1; dried active yeast, 1; brewers' years, 1; rye flour, 1; brown rice, 1; and alfalfa seeds,1. The occurrence of this pathogen in three types of soybean products should warrant further investigation of soybean derivatives as potentially significant sources of Salmonella.     

Usefulness of the stomacher in a microbiological regulatory laboratory.

The relative efficiency of the Waring blender, the Stomacher 400, and the Stomacher 3500 for preparing food samples for microbiological analysis was studied. Comparative aerobic plate count (APC) values were determined on 671 samples, representing 30 categories of foods. Of the 26 categories of nonfatty foods, the blender gave significantly higher geometric mean APC values than those given by the Stomacher 400 and the Stomacher 3500 in 65 and 69 percent of the categories, respectively. In a comparison of the two Stomacher models, the Stomacher 400 gave significantly higher geometric mean APC values than these given by the Stomacher 3500 in 73 percent of the food categories. Addition of Tween 80 to four categories of fatty foods at concentrations of 0.5, 1.0, and 2.0 percent did not raise the APC values given by either model of stomacher to the levels given by the Waring blender. Overall, the efficiency of both models of Stomacher, relative to the blender and to each other, was specific and depended upon the particular food being analyzed.     

Molecular drift of the bride of sevenless (boss) gene in Drosophila

DNA sequences were determined for three to five alleles of the bride-of- sevenless (boss) gene in each of four species of Drosophila. The product of boss is a transmembrane receptor for a ligand coded by the sevenless gene that triggers differentiation of the R7 photoreceptor cell in the compound eye. Population parameters affecting the rate and pattern of molecular evolution of boss were estimated from the multinomial configurations of nucleotide polymorphisms of synonymous codons. The time of divergence between D. melanogaster and D. simulans was estimated as approximately 1 Myr, that between D. teissieri and D. yakuba as approximately 0.75 Myr, and that between the two pairs of sibling species as approximately 2 Myr. (The boss genes themselves have estimated divergence times approximately 50% greater than the species divergence times.) The effective size of the species was estimated as approximately 5 x 10(6), and the average mutation rate was estimated as 1-2 x 10(-9)/nucleotide/generation. The ratio of amino acid polymorphisms within species to fixed differences between species suggests that approximately 25% of all possible single-step amino acid replacements in the boss gene product may be selectively neutral or nearly neutral. The data also imply that random genetic drift has been responsible for virtually all of the observed differences in the portion of the boss gene analyzed among the four species.      

High rate of DNA loss in the Drosophila melanogaster and Drosophila virilis species groups

We recently proposed that patterns of evolution of non-LTR retrotransposable elements can be used to study patterns of spontaneous mutation. Transposition of non-LTR retrotransposable elements commonly results in creation of 5' truncated, "dead-on-arrival" copies. These inactive copies are effectively pseudogenes and, according to the neutral theory, their molecular evolution ought to reflect rates and patterns of spontaneous mutation. Maximum parsimony can be used to separate the evolution of active lineages of a non-LTR element from the fate of the "dead-on-arrival" insertions and to directly assess the relative frequencies of different types of spontaneous mutations. We applied this approach using a non-LTR element, Helena, in the Drosophila virilis group and have demonstrated a surprisingly high incidence of large deletions and the virtual absence of insertions. Based on these results, we suggested that Drosophila in general may exhibit a high rate of spontaneous large deletions and have hypothesized that such a high rate of DNA loss may help to explain the puzzling dearth of bona fide pseudogenes in Drosophila. We also speculated that variation in the rate of spontaneous deletion may contribute to the divergence of genome size in different taxa by affecting the amount of superfluous "junk" DNA such as, for example, pseudogenes or long introns. In this paper, we extend our analysis to the D. melanogaster subgroup, which last shared a common ancestor with the D. virilis group approximately 40 MYA. In a different region of the same transposable element, Helena, we demonstrate that inactive copies accumulate deletions in species of the D. melanogaster subgroup at a rate very similar to that of the D. virilis group. These results strongly suggest that the high rate of DNA loss is a general feature of Drosophila and not a peculiar property of a particular stretch of DNA in a particular species group.      

The contractile basis of amoeboid movement: V. The control of gelation, solation, and contraction in extracts from dictyostelium discoideum

Motile extracts have been prepared from Dictyostelium discoideum by homogenization and differential centrifugation at 4 degrees C in a stabilization solution (60). These extracts gelled on warming to 25 degrees Celsius and contracted in response to micromolar Ca++ or a pH in excess of 7.0. Optimal gelation occurred in a solution containing 2.5 mM ethylene glycol-bis (β-aminoethyl ether)N,N,N',N'-tetraacetate (EGTA), 2.5 mM piperazine-N-N'-bis [2-ethane sulfonic acid] (PIPES), 1 mM MgC1(2), 1 mM ATP, and 20 mM KCI at ph 7.0 (relaxation solution), while micromolar levels of Ca++ inhibited gelation. Conditions that solated the gel elicited contraction of extracts containing myosin. This was true regardless of whether chemical (micromolar Ca++, pH >7.0, cytochalasin B, elevated concentrations of KCI, MgC1(2), and sucrose) or physical (pressure, mechanical stress, and cold) means were used to induce solation. Myosin was definitely required for contraction. During Ca++-or pH-elicited contraction: (a) actin, myosin, and a 95,000-dalton polypeptide were concentrated in the contracted extract; (b) the gelation activity was recovered in the material sqeezed out the contracting extract;(c) electron microscopy demonstrated that the number of free, recognizable F-actin filaments increased; (d) the actomyosin MgATPase activity was stimulated by 4- to 10-fold. In the absense of myosin the Dictyostelium extract did not contract, while gelation proceeded normally. During solation of the gel in the absense of myosin: (a) electron microscopy demonstrated that the number of free, recognizable F- actin filaments increased; (b) solation-dependent contraction of the extract and the Ca++-stimulated MgATPase activity were reconstituted by adding puried Dictyostelium myosin. Actin purified from the Dictyostelium extract did not gel (at 2 mg/ml), while low concentrations of actin (0.7-2 mg/ml) that contained several contaminating components underwent rapid Ca++ regulated gelation. These results indicated : (a) gelation in Dictyostelium extracts involves a specific Ca++-sensitive interaction between actin and several other components; (b) myosin is an absolute requirement for contraction of the extract; (c) actin-myosin interactions capable of producing force for movement are prevented in the gel, while solation of the gel by either physical or chemical means results in the release of F-actin capable of interaction with myosin and subsequent contraction. The effectiveness of physical agents in producting contraction suggests that the regulation of contraction by the gel is structural in nature.     

Distinct effects of SP-B and SP-C on the uptake of surfactant-like liposomes by alveolar cells in vivo and in vitro

The effects of surfactant protein B (SP-B) and SP-C on the uptake of surfactant-like liposomes by alveolar type II cells and alveolar macrophages were studied both in vivo and in vitro. In vivo, mechanically ventilated rats were intratracheally instilled with fluorescently labeled liposomes that had SP-B and/or SP-C incorporated in different concentrations. Consequently, the alveolar cells were isolated, and cell-associated fluorescence was determined using flow cytometry. The results show that the incorporation of SP-B does not influence the uptake, and it also does not in the presence of essential cofactors. The inclusion of SP-C in the liposomes enhanced the alveolar type II cells at a SP-C to lipid ratio of 2:100. If divalent cations (calcium and magnesium) were present at physiological concentrations in the liposome suspension, uptake of liposomes by alveolar macrophages was also enhanced. In vitro, the incorporation of SP-B affected uptake only at a protein-to-lipid ratio of 8:100, whereas the inclusion of SP-C in the liposomes leads to an increased uptake at a protein-to-lipid ratio of 1:100. From these results, it can be concluded that SP-B is unlikely to affect uptake of surfactant, whereas SP-C in combination with divalent cations and other solutes are capable of increasing the uptake.     

Saikosaponin-d,a novel SERCA inhibitor,induces autophagic cell death in apoptosis-defective cells

Autophagy is an important cellular process that controls cells in a normal homeostatic state by recycling nutrients to maintain cellular energy levels for cell survival via the turnover of proteins and damaged organelles. However, persistent activation of autophagy can lead to excessive depletion of cellular organelles and essential proteins, leading to caspase-independent autophagic cell death. As such, inducing cell death through this autophagic mechanism could be an alternative approach to the treatment of cancers. Recently, we have identified a novel autophagic inducer, saikosaponin-d (Ssd), from a medicinal plant that induces autophagy in various types of cancer cells through the formation of autophagosomes as measured by GFP-LC3 puncta formation. By computational virtual docking analysis, biochemical assays and advanced live-cell imaging techniques, Ssd was shown to increase cytosolic calcium level via direct inhibition of sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase pump, leading to autophagy induction through the activation of the Ca²⁺/calmodulin-dependent kinase kinase–AMP-activated protein kinase–mammalian target of rapamycin pathway. In addition, Ssd treatment causes the disruption of calcium homeostasis, which induces endoplasmic reticulum stress as well as the unfolded protein responses pathway. Ssd also proved to be a potent cytotoxic agent in apoptosis-defective or apoptosis-resistant mouse embryonic fibroblast cells, which either lack caspases 3, 7 or 8 or had the Bax-Bak double knockout. These results provide a detailed understanding of the mechanism of action of Ssd, as a novel autophagic inducer, which has the potential of being developed into an anti-cancer agent for targeting apoptosis-resistant cancer cells.     

In vivo and in vitro uptake of surfactant lipids by alveolar type II cells and macrophages

The uptake of fluorescent-labeled liposomes (with a surfactant-like composition) by alveolar macrophages and alveolar type II cells was studied using flow cytometry, in vivo by instillation of the labeled liposomes in the trachea of ventilated rats followed by isolation of the alveolar cells and determination of the cell-associated fluorescence, and in vitro by incubation of isolated alveolar cells with the fluorescent liposomes. The results show that the uptake of liposomes by the alveolar cells is time and concentration dependent. In vivo alveolar macrophages internalize more than three times as many liposomes as alveolar type II cells, whereas in vitro, the amount of internalized liposomes by these cells is approximately the same. In vitro, practically all the cells (70-75%) internalize liposomes, whereas in vivo only 30% of the alveolar type II cells ingest liposomes vs. 70% of the alveolar macrophages. These results indicate that in vivo, only a small subpopulation of alveolar type II cells is able to internalize surfactant liposomes.