Reactions of Hemoglobiniferous Cells to Aced and Basic Dyes under Varying Conditions of H-Ion Activity

1. An attempt has been made to apply Loeb's concept of the amphoteric nature of proteins for the discrimination of suspected hemoglobiniferous substances from known hemoglobiniferous substances according to their reactions to acid and basic dyes with reference to the isoelectric point of hemoglobin (pH 6.8).

2. The substances in the cytoplasm of known hemoglobiniferous cells (red blood corpuscles, normoblasts and erythroblasts) of the lymph nodes, spleen and bone marrow of the albino rat, when suspended in buffered dye-sucrose solutions, retain eosin on the acid side of pH 7.0, but the substances of the Russell bodies, suspected of being hemoglobiniferous, do not retain eosin at all; and the cytoplasm of the plasma cells, also alleged to be slightly hemoglobiniferous, only retains eosin on the acid side of pH 6.4.

3. The only basic dye used which did not precipitate in buffer solutions was methylene blue. This did not react with hemoglobin in accordance with Loeb's concept, because it did not penetrate mature red blood corpuscles and in those immature erythrocytes which it did penetrate it was precipitated by the reticulum.

4. Therefore, from the results obtained with the acid dye, it is tentatively concluded that the substance in the Russell bodies and in the cytoplasm of the plasma cells are not hemoglobiniferous because they do not react as do the substances in known hemoglobiniferous cells with reference to the isoelectric point of hemoglobin.

5. More investigation, however, must be carried out on both fresh and fixed material before a final unequivocal answer can be made to this problem.     

Reactions of Hemoglobiniferous Cells to Aced and Basic Dyes under Varying Conditions of H-Ion Activity

1. An attempt has been made to apply Loeb's concept of the amphoteric nature of proteins for the discrimination of suspected hemoglobiniferous substances from known hemoglobiniferous substances according to their reactions to acid and basic dyes with reference to the isoelectric point of hemoglobin (pH 6.8).

2. The substances in the cytoplasm of known hemoglobiniferous cells (red blood corpuscles, normoblasts and erythroblasts) of the lymph nodes, spleen and bone marrow of the albino rat, when suspended in buffered dye-sucrose solutions, retain eosin on the acid side of pH 7.0, but the substances of the Russell bodies, suspected of being hemoglobiniferous, do not retain eosin at all; and the cytoplasm of the plasma cells, also alleged to be slightly hemoglobiniferous, only retains eosin on the acid side of pH 6.4.

3. The only basic dye used which did not precipitate in buffer solutions was methylene blue. This did not react with hemoglobin in accordance with Loeb's concept, because it did not penetrate mature red blood corpuscles and in those immature erythrocytes which it did penetrate it was precipitated by the reticulum.

4. Therefore, from the results obtained with the acid dye, it is tentatively concluded that the substance in the Russell bodies and in the cytoplasm of the plasma cells are not hemoglobiniferous because they do not react as do the substances in known hemoglobiniferous cells with reference to the isoelectric point of hemoglobin.

5. More investigation, however, must be carried out on both fresh and fixed material before a final unequivocal answer can be made to this problem.     

Antimicrobial peptides from the venoms of Vespa bicolor Fabricius

Hornets possess highly toxic venoms, which are rich in toxins, enzymes and biologically active peptides. Many bioactive substances have been identified from wasp venoms. Vespa mastoparan (MP-VBs) and Vespa chemotatic peptide presenting antimicrobial action (VESP-VBs) were purified and characterized from the venom of the wasp, Vespa bicolor Fabricius. The precursors encoding VESP-VBs and MP-VBs were cloned from the cDNA library of the venomous glands. Analyzed by FAB-MS, the amino acid sequence and molecular mass for VESP-VB1 were FMPIIGRLMSGSL and 1420.6, for MP-VB1 were INMKASAAVAKKLL and 1456.5, respectively. The primary structures of these peptides are homologous to those of chemotactic peptides and mastoparans isolated from other vespid venoms. These peptides showed strong antimicrobial activities against bacteria and fungi and induced mast cell degranulation, but displayed almost no hemolytic activity towards human blood red cells.     

Correlation between simulated physicochemical properties and hemolycity of protegrin-like antimicrobial peptides: predicting experimental toxicity

The therapeutic, antibiotic potential of antimicrobial peptides can be prohibitively diminished because of the cytotoxicity and hemolytic profiles they exhibit. Quantifying and predicting antimicrobial peptide toxicity against host cells is thus an important goal of AMP related research. In this work, we present quantitative structure activity relationships for toxicity of protegrin-like antimicrobial peptides against human cells (epithelial and red blood cells) based on physicochemical properties, such as interaction energies and radius of gyration, calculated from molecular dynamics simulations of the peptides in aqueous solvent. The hypothesis is that physicochemical properties of peptides, as manifest by their structure and interactions in a solvent and as captured by atomistic simulations, are responsible for their toxicity against human cells. Protegrins are beta-hairpin peptides with high activity against a wide variety of microbial species, but in their native state are toxic to human cells. Sixty peptides with experimentally determined toxicities were used to develop the models. We test the resulting relationships to determine their ability to predict the toxicity of several protegrin-like peptides. The developed QSARs provide insight into the mechanism of cytotoxic action of antimicrobial peptides. In a subsequent blind test, the QSAR correctly ranked four of five protegrin analogues newly synthesized and tested for toxicity.     

Micro-scission of YAC tumor cells during antibody-dependent cellular cytotoxicity mediated by human neutrophils

The cellular events accompanying neutrophil-mediated antibody-dependent cellular cytotoxicity (ADCC) directed against YAC erythroleukemic target cells have been studied by time-lapse fluorescence-intensified microscopy. The YAC plasma membrane and cytosol were labeled with the fluorescent probes diC18Icc and eosin Y, respectively. Fluorescently labeled and IgG-opsonized YAC cells were incubated at 37 degrees C while observed by optical microscopy. During temporal studies of neutrophil-YAC conjugates, the cytosol of YAC cells accumulated in tubular and spherical compartments of the neutrophils' vacuolar apparatuses. To distinguish between several possible mechanisms of target cytosol uptake, diC18Icc-labeled YAC cells were observed during identical conditions. The membrane label diC18Icc was found to accumulate within neutrophils in an identical fashion. At roughly 30 min, 25 and 38% of neutrophils in apparent conjugates had internalized tumor cell cytosol or plasma membrane, respectively, within a vesicular compartment. The IgG-dependent uptake of eosin Y and diC18Icc by neutrophils was diminished by exposure to 2.5 mM sodium azide. When cells were exposed to 5.5 mM sodium azide, 1 mM iodoacetamide, or 4 degrees C, conjugate formation and uptake of eosin Y or diC18Icc were abolished. An artifactual accumulation of eosin Y or diC18Icc in neutrophils was further ruled out by control studies. Non-specific exchanges of eosin Y and diC18Icc labels of YAC cells with tannic acid-treated red blood cells (RBCs) and normal neutrophils were studied. Since hemoglobin binds tightly to eosin Y, RBCs can easily detect eosin Y leakage. No exchange of eosin Y or diC18Icc from YAC cells into bound tannic acid-treated erythrocytes was found.(ABSTRACT TRUNCATED AT 250 WORDS)     

THE EFFECT OF HEMOLYTIC SUBSTANCES ON WHITE CELL RESPIRATION

Substances such as saponin, the bile salts, etc., which produce lysis of red cells also produce cytolysis of white cells from rabbit peritoneal exudates, the arbitrary criterion of their cytolytic effect being their ability to depress the O₂ consumption of the leucocytes. The amount of cytolysis increases regularly as the amount of the added lysin is increased, and sufficiently large quantities of saponin, sodium taurocholate, sodium glycocholate, or sodium oleate are capable of virtually abolishing the O₂ consumption altogether. At the same time, it can be shown that a lysin such as saponin is used up in combining with the white cells in much the same way as it is used up in combining with red cells, and the reduction in oxygen consumption appears to be roughly proportional to the amount so combined. The action of these lytic substances on white cells, in fact, is very similar to their action on red cells, due allowance being made for the fact that the cytolysis of the white cell is probably not an all-or-none process like hemolysis. White cell respiration is also depressed in hypotonic solutions, the respiration being virtually linear with the tonicity.     

On the action of sulfanilamide

Summary A sulfanilamide activating principle was found to be present in red cells of the horse. This activator substance is active in the rather high dilution of 0.5% haemolysed red cells.The substance or substances are present in the red cells, not in their cell membranes. They seem to be of a protein nature or adsorbed to the protein (haemoglobin).In some media no sulfanilamide action is obtained without the activator. In other media sulfanilamide action, though clearly present, is markedly enhanced. So it must be emphasized, that the substance under discussion is an activator and not a conditio sine qua non for the sulfanilamide action and its characteristics.The substance is activating sulfanilamide against streptococci, staphylococci andB. coli.The substance is not present in human blood or in the red cells of sheep, rabbits or mice.Sixth communication: K. C.Winkler, Antonie van Leeuwenhoek8, 10, 1942.     

A study of toxic substances in pertussis cultures]

For the purpose of studying the toxic properties of pertussis strains with different agglutination composition and to ascertain the interrelationship between the action of the toxic substances and the serological type of the strains the author used a test of the weight change in albino mice to which crude and heated (at 56 degrees C for 10 minutes) suspensions of the strains of various serological types were injected intraperitoneally. The toxic properties were checked in 17 strains. The test of the change of the animal body weight with the determination of the regression coefficient permitted to determine roughly the presence of the toxic substances in the strains; the action of the thermostable dermonecrotic toxin and thermostable endotoxin was expressed with greater constancy than that of the lymphocytosis stimulating factor. There was no interrelationship between the manifestation of the action of toxic substances in the pertussis strains and their serological type. The toxic activity peristed in the strains stored in dried condition.     

Toxicity of para-aminobenzhydrazide and its influence on nucleic acid biosynthesis in cultures of normal and tumor cells

We have investigated cytotoxic action of p-aminobenzhydrazide and its influence on biosynthesis of nucleic acids in cultures of intact cells, tumor cells and intact cells stimulated by phytohemagglutinin. p-Aminobenzhydrazide is considered as a representative of hydrazine's derivatives (in particular, of hydrazine sulphate). We compare its action with that of a typical cytotoxic agent such as iododeoxyuridine. We have found that p-aminobenzhydrazide influences biosynthesis of nucleic acids in the same way as iododeoxyuridine. However it acts toxically on tumor cells though it is not toxic for intact cells so that its action is different as compared to that of cytotoxic agents. Specific toxic action of aminobenzhydrazide on tumor cells may be due to the enhancement of antitumor activity substances of this compound and absence of such enhancement of side toxic effects.     

THE KINETICS OF IN VIVO HEMOLYTIC SYSTEMS

This paper is concerned with a variety of questions which bear on the occurrence of hemolysis in vivo, and with the possibility of regarding the contents of the blood stream as a hemolytic system in which a steady state is maintained by the production of new red cells to replace those which are destroyed. The material which is dealt with includes the following. 1. Mixtures of Lysins, Accelerators, and Inhibitors.—The effects of individual accelerators and inhibitors in mixtures, like the effects of individual lysins, are roughly additive in simple systems, the acceleration or inhibition produced by the individual substances being most conveniently measured in terms of R-values. 2. Normal Intravascular Lysins.—These probably play only a small part in red cell destruction unless their concentration rises to unusual levels, or unless their effects are enhanced by accelerators, or by the reduction of the concentration of normal inhibitors. The three normal in vivo hemolytic processes for which there is substantial evidence involve (a) the action of the bile salts and of the soaps derived from chyle, (b) the action of the spleen, and (c) the action of hemolytic substances derived from tissues. The recent observations of Maegraith, Findlay, and Martin on the presence of widely distributed tissue lysins are confirmed except for their conclusion that these lysins are species-specific. Species-specific tissue lysins, if present, are not the only lysins derivable from tissues by simple immersion in saline, for non-species-specific lytic substances can also be obtained, and seem to be similar to the "lysolecithin" which some regard as responsible for the action of the spleen on red cell fragility and shape. 3. Plasma Inhibitors.—About 30 per cent of the total inhibitory effect of plasma for saponin hemolysis is due to the contained cholesterol, while 25 per cent at most is due to the plasma proteins, particularly globulins. The remaining 45 per cent is probably accounted for by enhancing effects among the inhibitors; e.g., the enhancing effect of lecithin on the cholesterol inhibition. The mechanism of the inhibition is still incompletely understood; probably reactions between inhibitor and lysin and reactions between inhibitor and components of the red cell surface are both involved, and it is important to observe that the inhibitory effect of plasma or of a plasma constituent may be greater in systems containing one lysin than in systems containing another. No evidence for diffusible inhibitory substances in plasma has been found, and the variations observed in the inhibitory power of human plasma seem to be related to the combined concentrations of cholesterol, protein, and probably lecithin, rather than to the cholesterol content alone. For this reason the inhibitory power tends to be low under conditions of poor nutrition. 4. The Steady State and the Kinetics of Hemolysis In Vivo.—On the assumption that the steady state is the result of a balance between a process which produces red cells and a process which destroys them, equations have been developed for the way in which cells of different resistances are affected when the rate of destruction changes. A method for analyzing experimental curves is described and illustrated. In general, this part of the paper relates the level of the red cell count in the animal to the intensity of the hemolytic processes taking place in vivo, and does not lend itself to detailed abstraction.     

A New Toxic Substance,Teleocidin, Produced by Streptomyces.

In the course of studies on the screening of specific toxic substances produced by Streptomyces against aquatic organisms, it was recognised that a newly isolated Streptmyces, 2A 1563, which was considered to be a variant species of Streptomyces mediocidicus produces a new toxic substance, Teleocidin. Teleocidin was isolated as a white powder resembling crystals with a crystal appearance from the methanol extracts of the cultured mycellium, and showed a specific toxic action toward Japanese killifish and mice, but did not exhibit any inhibition against microorganisms.     

Endocrine disruptors: Revisiting concepts and dogma in toxicology

During the last decades, a large number of observations have shown that some exogenous substances could interfere with hormone levels or hormone action and could induce toxic effects. This has led to the identification of endocrine disruptors more than 25 years ago as a new class of toxic agents (Zoeller et al., 2014). Those widely used agents correspond to a variety of chemical classes, are not identified by their chemical structure or by a specific type of usage, but rather by their mechanisms of action; this is not unprecedented in toxicology since genotoxicants have also been identified by their mechanism of action, i.e. their ability to alter DNA structure and function.     

Neuroprotection: a comparative view of vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide

The neuroprotective properties of vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) place these peptides in a special category of ligands that have implications for our understanding of pathological conditions as well as a potential basis for therapeutic intervention. It is remarkable that these peptides have a protective impact against such a wide variety of clinical relevant toxic substances. This protective diversity is consistent with the multiple pathways that are activated or inhibited by the action of these peptides. Although knowledge is emerging on the neuroprotective mechanisms of VIP and PACAP, it is already evident that these two peptides are not identical in their action and each peptide has multiple mechanisms that allow for neuroprotective diversity. The multiple intracellular signaling pathways and differing extracellular mediators of neuroprotection contribute to this diversity of action. In this review, examples of neuroprotective actions will be presented that serve to demonstrate the remarkable breadth of neuroprotective processes produced by VIP and PACAP.     

Transfer of band 3, the erythrocyte anion transporter, between phospholipid vesicles and cells

Band 3, the anion transport protein of human erythrocyte membranes, can be transferred from cells to liposomes and from liposomes back to cell membranes, retaining function and native orientation. After incubation with cells, sonicated phosphatidylcholine vesicles bind a transmembrane protein that comigrates with band 3 on sodium dodecyl sulfate-polyacrylamide gels. Like native red cell band 3, the vesicle-bound protein is cleaved by chymotrypsin into 65- and 30-kdalton fragments and is not cleaved by trypsin. The protein can be cross-linked by copper-phenanthroline oxidation either before or after transfer to vesicles; in either case, the vesicle fractions contain high molecular weight material that is dissociated into 95-kdalton species by mercaptoethanol. Band 3-vesicle complexes contain no detectable cell lipid and are specifically permeable to anions. Greater than 99% of their anion uptake can be blocked by the band 3 inhibitor 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS). Red cells whose band 3 function has been blocked irreversibly by DIDS or eosin maleimide regain part of their anion permeability upon incubation with band 3-vesicle complexes. Under the conditions employed, an average of one copy of functional band 3 is delivered to half of the cells, increasing by 2.3-fold the number of cells containing functional anion transporters. Incubation of pure lipid vesicles or red cell membrane buds with either normal red cells or eosin maleimide inhibited cells has no detectable effect on the cells' anion permeability.     

Cell Response to Surface Stimulation

The injection of anti-D into Rh-negative subjects who have Rh-positive red cells in the circulation results in the inhibition of immunization against the D-antigen¹. On the other hand, subjects who have had a primary anti-D response to Rh-positive red cells frequently give a good secondary response to small doses of red cells despite the presence of anti-D in their plasma. The difference in action between the passively-administered and the actively-produced anti-D might lie in the fact that the injected IgG anti-D is derived from a pool of donors and therefore contains a number of IgG antigens which are foreign to the recipient, compared with the autologous nature of the anti-D present after a primary response.     

Patterns in the production of antiherbivore chemical defenses in plant communities

The diversity of chemical identity and mode of action of toxic substances present in plants is suggested to be partially the result of generalist herbivore grazing pressure, while convergence in digestibility-reducing substances and their method of function is due primarily to specialist herbivore. Analysis of alkaloid patterns from herbaceous and woody perennials show that most species differ in their alkaloid composition and many sympatric species and subspecies show unique patterns. The high degree of functional convergence in digestibility-reducing substances is supported by convergent patterns in tannins among woody perennials.     

A new simple screening method for the detection of cytotoxic substances produced by harmful red tide phytoplankton

A highly sensitive, but simple and quantitative, cytotoxic assay method for the detection of toxic substances produced by red tide phytoplankton was developed by utilizing Vero cells which were the most resistant to seawater among the six cell lines tested. Heterocapsa circularisquama, which is known to be highly toxic to shellfish, showed cytotoxicity to Vero cells in a cell-density dependent manner when Vero cells were directly exposed to the cell suspension of H. circularisquama in seawater-based plankton culture medium, whereas Heterocapsa triquetra, which is morphologically similar to H. circularisquama but non-toxic to shellfish, showed no cytotoxic effect. Since the potent cytotoxicity was also detected in the cell-free culture supernatant of H. circularisquama, it was suggested that a certain cytotoxic substance is extracellularly secreted by H. circularisquama. Furthermore, by this direct exposure method, we found that Alexandrium fraterculus, Alexandrium tamiyavanichii, Alexandrium tamarense, and Alexandrium affine but not Alexandrium taylorii and Alexandrium catenella cause toxic effect on Vero cells with different extent depending on species. By gel-filtration and subsequent two cytotoxicity assays using Vero and mouse neuroblastoma cell line (Neuro-2a), we found that high molecular weight cytotoxic substance distinct from paralytic shellfish poisoning toxins is present in the aqueous extract of A. tamarense. These results suggest that our 96-well microplate cytotoxicity assay using Vero cells is useful not only as a primary screening assay for the detection of potential toxic activity of harmful phytoplankton but also as a quantitative routine toxicity assay for following the active substances during the extraction and purification processes.     

Selective Recognition of Various Erythrocytes in Endocytosis by Mouse Peritoneal Macrophages

LITTLE is known about the mechanism by which antibodies are produced against foreign substances but not against components of the body. Mishell and Dutton¹ developed a cell culture system for formation of antibody against sheep red blood cells (RBC) as antigen¹. Mosier demonstrated that both macrophages and lymphocytes in mouse spleen are responsible for antibody formation².     

Cytotoxic action of the chemical substances found as admixtures in medical immunobiological preparations

The cytotoxic action (CTA) of chemical substances contained as admixtures in medical immunobiological preparations on human diploid cells has been studied. Such chemical substances as rivanol and merthiolate in admissible concentrations show the highest degree of CTA. The results obtained in this investigation indicate that different concentrations of chemical substances may produce equal CTA; thus, thiolate in toxic in a dose of 0.8 microgram/ml; the same CTA is produced by aluminium sulfate in a concentration of 500 micrograms/ml. Small doses of chemical substances, producing no explicit manifestations of the cytotoxic effect, may have latent CTA determined by additional methods of investigation. CTA may be manifested as lethal, sublethal and latent cell lesions. In working out regulations on the test for CTA it is expedient to indicate admissible residual amounts of chemical substances contained in finished medical immunobiological preparations, considering that these amounts must be incapable of producing CTA in cell cultures. The conclusion has been made on the expediency of denoting small amounts of chemical substances capable of producing latent CTA as tentatively tolerable doses.     

Characterization of microalgae and associated bacteria collected from shellfish harvesting areas

Marine algal toxins are an important cause of seafood-associated outbreaks. Some marine bacteria living in association with algae are able to produce channel-blocking substances similar to PSP and TTX toxins and a role of these bacteria in the toxicity of dinoflagellates has been hypothesized. The aim of this study was to monitor, over a period of 2 years, areas used in shellfish production in the northern Adriatic Sea, through the determination of phytoplankton and the characterization of bacteria isolated from algae. Toxicity tests on bacterial extracts were performed using in vivo (mouse) and in vitro (cell culture) tests and by HPLC. The Dinophysis genus was detected throughout the year, while the Alexandrium genus was present in winter and spring. Sixteen bacteria isolated from algae, out of 61 bacterial strains tested by in vitro assay, were found to be producers of toxic substances that could block sodium channels in cells. HPLC analysis for the detection of PSP and TTX toxins always gave negative results, but their presence in concentrations undetectable by HPLC, and/or the production of chemically different substances with similar biological action, could not be excluded.