Effects of the methanol extract residue (MER) tubercle bacillus fraction on the production of antibodies in vitro. II. Effects on macrophage and lymphocyte populations

The effect of the methanol extract residue (MER) fraction of BCG tubercle bacilli on the generation of primary antibody responsiveness in vitro to sheep red blood cells (SRBC) was ascertained in cell reconstitution experiments, employing enriched populations of mouse macrophages and of T and B lymphocytes. In each of the antibody generation cultures one or another of the cell fractions had been exposed to MER, either by treatment of the donor animals or by preincubation with the agent for 48 hr in vitro. In some experiments, supernatants of MER-preincubated cells were employed in place of the cells. Macrophages and T cells that had been exposed to MER in vivo or in vitro and their supernatants demonstrated a markedly greater effect than nonexposed cells in the generation of direct specific plaque-forming cells (PFC) upon antigenic stimulation of the cultures with SRBC. In contrast, PFC production was not stimulated in B-lymphocyte populations that had been in contact with the agent.     

Effects of chemical modification of carboxyl groups on the voltage-clamped nerve fiber of the frog

Summary Voltage-clamped single nerve fibers of the frogRana esculenta were treated with the carboxyl group activating reagent N-ethoxy-carbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) in the presence of different primary amines and without added amine. Carboxyl groups form stable amide bonds with primary amines in the presence of EEDQ. EEDQ treatment reduced the sodium current considerably and irreversibly, regardless of the presence of a primary amine in the Ringer's solution. The potassium current was also reduced. After modification the reduced sodium currents inactivated slowly and incompletely. The descending branch of the sodium current-voltage relation,I _Na(E), was shifted along the voltage axis in the depolarizing direction. The size of the shift was strongly dependent on the amine present during modification with EEDQ. The voltage-dependence of sodium inactivation,h _x (E), was shifted to more positive values of membrane potential by EEDQ in the presence of ethylenediamine (11 mV) and glucosamine (3 mV). In contrast, a small shift to more negative potentials occurred in the presence of taurine (–3 mV) or without the addition of an amine (–2 mV). A tenfold increase of the calcium concentration still shifted theI _Na(E) andh _x (E) curves of the chemically modified fibers. However, these shifts were smaller than those observed on untreated fibers. The currents remaining after the modification were completely blocked by tetrodotoxin; no change of the reversal potential occurred.     

The development of a technique for the morphometric analysis of invasion in cancer

We describe tests of the feasibility of a reconstructive technique to discriminate between expansive growth and active cell movement in the invasion of tissues by cancer cells. The densities of cancer cells in 2210 microns2 (grid) squares of standard 6 microns fixed, stained histologic sections of a nodule and an invasive cutaneous melanoma were determined, and density maps of the tumors constructed. An abrupt transition from saturation density to zero cell density was observed at the advancing edge (towards the stratum corneum) of the tumor nodule which was consistent with a model for expansion by growth (vis a tergo). In contrast, at the advancing edge of the invasive tumor, the transition from saturation to zero density (towards the subcutaneous tissues) occurred more gradually, over approximately 400 mum, which was consistent with a model for invasion by active movement of melanoma cells. The occurrence of statistically significant "high density regions" near to the advancing edge of the invasive tumor is consistent with an invasive pattern of active movement followed by focal proliferation of the cancer cells, in a repetitious manner. It therefore appears feasible to make kinetic reconstructions of some of the events in invasion, from static quantitative observations.     

The role of T3 surface molecules in the activation of human T cells: a two-stimulus requirement for IL 2 production reflects events occurring at a pre-translational level

The human T cell leukemia Jurkat was used as a model to examine the requirements of T cell activation. These studies demonstrated that antibodies reactive with the T cell-specific T3 antigen were insufficient to result in the activation of Jurkat cells, determined by the secretion of IL 2. IL 2 production occurred only in the presence of a second stimulus, the phorbol ester PMA. With the use of an IL 2-specific cDNA probe, the appearance of IL 2 RNA, similarly, occurred only when cells were stimulated with both anti-T3 antibodies and PMA. These results demonstrate a two-stimulus requirement for gene expression in human T cells.     

Operant behavior in transition reflects neonatal exposure to cadmium

Male Long-Evans rats were injected with 0, 1, 3, or 6 mg/kg of cadmium chloride on the first day of life. Animals free of morphological stigmata at weaning were selected for study. Tissue concentrations of cadmium and operant behavior under various fixed-ratio (FR) schedules of reinforcement were evaluated when these rats were adults. Dose-related increases in cadmium were present in the brains, livers, and kidneys. Dose-related differences in behavior were most evident during the transition from fixed ratio 25 (FR 25 or 25 responses/reinforcer) to FR 75. An inverted U describes the relationship between response output during the transition to FR 75 and cadmium chloride dose response output increased at 3 mg/kg and decreased at 6 mg/kg. The rate decreases were not correlated with weight loss that appeared after some of the animals exposed to 6 mg/kg reached 60 days of age. Challenge doses of d-amphetamine revealed no interaction between neonatal exposure to cadmium and d-amphetamine. The occurrence of alterations in operant behavior in animals that appeared normal on a number of preweaning evaluations suggests that operant behavior in transition was sensitive to subtle effects not observed with other commonly used tests. The data provide evidence for delayed effects in the adult that are due to neonatal exposure to cadmium.     

Computer-aided three-dimensional reconstructions of the arrangement of primary spermatocytes in human seminiferous tubules

Summary With the use of a digital image-processing method three-dimensional reconstructions of the arrangement of spermatocytes in human seminiferous tubules were performed. With this method it was possible to investigate the cellular distribution in the tubule in nearly any given perspective and projection. In addition, by means of simple mathematical procedures, such as by transformation of Cartesian coordinates into cylindrical coordinates, it was possible to vary the shape of a reconstruction, i.e., to convert the cylindrical image of a tubular portion into a right-angled r--z-representation.The present work not only confirms the existence of a complex helical plan of organization of the human seminiferous epithelium but also provides further aspects of the phenomenon of physiological germ-cell loss and its integration into the kinetics of spermatogenesis.Dedicated to Prof. E.C. Roosen-Runge, Seattle, on the occasion of his 75th birthday     

Plasmodium berghei: a mouse model for the "sudden death" and "malarial lung" syndromes

A mouse model for the "sudden death" and "malarial lung" syndromes is described. Mice of the C3H/z strain succumb suddenly approximately 7 days after an infection with Plasmodium berghei becomes patent, at a time when parasitemia is still moderate (6 to 8%). Death could be shown to be due to anaphylactoid shock, probably induced by soluble immune complexes. Increased vascular permeability caused transudation and leakage of serum proteins into the interstitium and the alveoli. The lungs were found to be edematous, with a fine granular precipitate in the alveoli and adherent to the vascular walls. The precipitates reacted with antiglobulins G and M, and could be shown to also contain malaria antigens and C3/4. A dramatic drop in hematocrit was recorded several hours before death, indicating the sudden release of malaria antigens. The myocardium of animals that had died very suddenly showed a patchy loss of phosphorylase activity. This loss of activity was much more extensive, and sometimes almost total, when there had been an agonal period of several (1 to 3) hours before death. In these cases the irreversibility of the myocardial damage was also indicated by the loss of activity of the dehydrogenases, as well as by typical inflammatory reactions of granulocytic and histiocytic infiltrations. The hearts thus presented a typical picture of the acute and peracute shock syndromes. In acute shock cardiac insufficiency develops so suddenly that death ensues before irreversible damage has occurred, and cardiac insufficiency can only be demonstrated by the most sensitive of enzyme histochemical means. In the present case shock was induced by the anaphylactoid activity of immune complexes with the lung as target organ. The described syndrome appears analogous to human "malarial lung."     

Biphasic culture system for rapid Campylobacter cultivation.

We developed a biphasic culture system consisting of 4 ml of brucella agar (BA) and 6 ml of brucella broth (BB) in 25-cm2 tissue culture flasks, which were incubated in air (BB/BAa) or in a gas mixture of 5% O2, 10% CO2, and 85% N2 (BB/BAg). These media were also used with a supplement consisting of ferrous sulfate, sodium metabisulfite, and sodium pyruvate and incubated as above (FB/FAa and FB/FAg, respectively). Highly satisfactory growth of Campylobacter jejuni 301 was obtained with all medium-gas phase combinations provided that the number of viable cells in the inoculum was large (greater than or equal to 10(6)/ml). The use of FB/FAa permitted the inoculum to be reduced to 100 cells per ml. With an adjusted gas phase (BB/BAg and FB/FAg), near-optimal growth was obtained from an inoculum of 1 to 10 cells per ml. Under most of these conditions the generation time was approximately 90 min. During the logarithmic growth phase, the cells retained their typical spiral morphology and high motility. These media also proved to be highly satisfactory for the cultivation of fresh isolates as well as other stock strains of Campylobacter. When the broth phase of the cultures, after addition of 15% glycerol, was quickly frozen and maintained at -70 degrees C, all strains thus far examined were readily recoverable and satisfactorily cultivated without additional passage.     

Tissue-specific expression of the rat glutathione S-transferases

Tissue-specific patterns of rat glutathione S-transferase expression have been demonstrated by in vitro translation of purified poly(A) RNAs and by protein purification. Poly(A) RNAs from six rat tissues including heart, kidney, liver, lung, spleen, and testis were used to program in vitro translation with the rabbit reticulocyte lysate system and [35S]methionine. The glutathione S-transferase subunits synthesized in vitro were purified from the translation products by affinity chromatography on S-hexylglutathione-linked Sepharose 6B columns. The affinity bound fractions were analyzed by Na dodecyl SO4-polyacrylamide gel electrophoresis and fluorography. A subunit of Mr = 22,000 detected in the in vitro translation products of poly(A) RNAs from heart, kidney, lung, spleen, and testis is missing from the translation products of liver poly(A) RNAs. This Mr = 22,000 subunit is present only in the anionic glutathione S-transferase fraction purified from rat heart, kidney, lung, spleen, and testis. Purified anionic glutathione S-transferase from rat liver does not contain this subunit. The relative specific activities toward a dozen different substrates also demonstrate the nonidentity between liver and kidney anionic glutathione S-transferases. In addition, among the glutathione S-transferase subunits expressed in the liver, some of them could not be detected in the other tissues investigated. Our results indicate that tissue-specific expression of rat glutathione S-transferases may occur pretranslationally.