Isolation and characterization of the mycobacterial phagosome: segregation from the endosomal/lysosomal pathway

Mycobacteria have the ability to persist within host phagocytes, and their success as intracellular pathogens is thought to be related to the ability to modify their intracellular environment. After entry into phagocytes, mycobacteria-containing phagosomes acquire markers for the endosomal pathway, but do not fuse with lysosomes. The molecular machinery that is involved in the entry and survival of mycobacteria in host cells is poorly characterized. Here we describe the use of organelle electrophoresis to study the uptake of Mycobacterium bovis bacille Calmette Guerin (BCG) into murine macrophages. We demonstrate that live, but not dead, mycobacteria occupy a phagosome that can be physically separated from endosomal/lysosomal compartments. Biochemical analysis of purified mycobacterial phagosomes revealed the absence of endosomal/lysosomal markers LAMP-1 and β-hexosaminidase. Combining subcellular fractionation with two-dimensional gel electrophoresis, we found that a set of host proteins was present in phagosomes that were absent from endosomal/lysosomal compartments. The residence of mycobacteria in compartments outside the endosomal/lysosomal system may explain their persistence inside host cells and their sequestration from immune recognition. Furthermore, the approach described here may contribute to an improved understanding of the molecular mechanisms that determine the intracellular fate of mycobacteria during infection.     

Detection of oligoclonal T lymphocytes in lymph nodes draining from advanced non-small-cell lung cancer

Despite the combined use of surgery and chemoradiotherapy, the poor prognosis of advanced non-smallcell lung cancer (NSCLC) requires the definition of new therapeutic approaches. The presence of T lymphocytes, with peculiar phenotypic, functional and molecular characteristics within the tumour, suggested the possible use of these cells, expanded in vitro, in protocols of adoptive immunotherapy. We have described how a population of oligoclonal T lymphocytes, derived from advanced NSCLC, can be expanded in vitro and has the capability of lysing autologous cancer cells. What is more important, we observed that patients with advanced NSCLC, treated with TIL expanded in vitro and recombinant interleukin-2, seemed to have a disease-free period longer than that of patients treated with conventional chemoradiotherapy. in an attempt to find new sources of specific lymphocytes for immunotherapy, we describe the analysis of the phenotypic, functional and molecular characteristics of T lymphocytes, derived from lymph nodes draining advanced NSCLC. In this paper we show that these cells, have restriction patterns of T cell receptor chain similar to those detectable in the population of infiltrating T lymphocytes. This finding suggests that T cells derived from draining lymph nodes of advanced NSCLC have peculiar characteristics and can be a suitable source of effector cells for protocols of adoptive immunotherapy in lung cancer treatment.     

Seasonal changes in the number of neurocytes containing lipofuscin pigment in selected areas of the brain of Rana temporaria L

The number of neurocytes containing lipofuscin pigment (LEP) was determined in the nucleus infundibularis dorsalis (NID), the subfornical organ (SFO) and the medulla oblongata (MO) of the brain of sexually mature females and males of Rana temporaria L. approximately identical in length and age. The frogs were captured in their natural habitat in seven periods of the annual cycle. The number of LFP-positive neurocytes shows distinct seasonal variations in the analyzed areas. In general, neurocytes containing LFP were found in higher numbers during the winter months, and their significant drop was observed in summer. The results seem to suggest that cells containing LFP represent a certain kind of dynamic cellular system responding to various internal and/or external factors changing within a year.     

Influence of actinomycin-D on the diurnal rhythm of cytoplasmic RNA in the neurocytes of selected nuclei of mouse hypothalamus

The influence of a single dose of actinomycin-D (1 mg/kg body weight) on the diurnal rhythm of cytoplasmic RNA was studied in neurocytes of the supraoptic nucleus (SON), paraventricular nucleus (PVN), and arcuate nucleus (AN) by microspectrophotometer. Cosinor analysis of the obtained results revealed a disturbed diurnal rhythm of cytoplasmic RNA in the animals treated with actinomycin-D. The mean diurnal RNA content in the SON, PVN, and AN neurocytes decreased, the amplitude of diurnal fluctuation was reduced and the acrophase was shifted to earlier hours.     

Haptoglobin and lipoperoxides in rabbit plasma under turpentine- and endotoxin-induced inflammation

Inflammation was induced in rabbits by injection of turpentine or/and endotoxin. Haptoglobin (Hp) and lipoperoxide (as malondialdehyde - MDA) concentrations in rabbit plasma, were measured. In some experiments indomethacin was administered, as a specific control of anti-inflammatory action. In the course of the reagents-induced inflammation there existed positive correlation between changes in Hp and MDA levels. Indomethacin was found to eliminate this effect. Under the model system of experiments, Hp as presumed endogenous inhibitor of prostaglandin synthesis, even in concentrations in rabbits' blood 15-20 times higher than normal, did not exert any protective action against inflammation.     

Diurnal rhythm of hypophyseal cells in mich. II. Pars intermedia.

By the use of Mac Conaill's lead hematoxylin, periodic acid and Schiff's reagent (PAS, PbH-positive and PAS-positive cells were distinguished in the pars intermedia of the hypophysis in mice. Nuclear volume in PbH-positive and PAS-positive cells in the pars intermedia of the hypophysis in male and female mice under conditions of 12 h light and 12 h darkness shows distinct diurnal rhythmicity. Maximum nuclear volume in PbH-positive cells of the pars intermedia in both sexes was observed at 1800 h, and minimum at 2400 h. In the Pas-positive cells in females maximum nuclear volume was observed at 600 h, and minimum at 2400 h. In males maximal nuclear volume in these cells appears at 2400 h, and minimum at 1800 h. Maximum number of vacuoles in the nuclei of PbH-positive cells in the pars intermedia in both sexes appeared at 1800 h, and minimum at 2400 h. Maximum numbers of vacuoles in the nuclei of PAS-positive cells in females was noted at 1200 h, and minimum at 2400 h. In males the maximum number of vacuoles appeared at 600 h, and minimum at 1200 h. Differences in the number of vacuoles in the nuclei of PAS-positive cells between males and females were also noted.     

Phytochrome-mediated control of grana and stroma thylakoid formation in plastids of mustard cotyledons

The etioplast»chloroplast transition in the cotyledons of mustard seedlings (Sinapis alba L.) has been studied by electron microscopy. It was found that the active form of phytochrome, established by a red light pulse pretreatment, increases the initial rate and eliminates the lag of grana and stroma thylakoid formation after the onset of white light 60 h after sowing. The effect of a pretreatment with 15 s red light pulses is fully reversible by 756 nm light pulses. This reversibility is lost within 5 min. Evidence is presented which suggests that the time course of grana and stroma thylakoid formation is not correlated with the time course of the dispersal of the prolamellar body. The different functions of phytochrome and chlorophyll in controlling thylakoid formation are discussed.     

Influence of chronic doses of mercuric acetate and lead acetate on the number and activity of Gomori-positive glial cells in the mouse brain.

Intraperitoneal injections of mercuric acetate or lead acetate in doses of 0.2 mg daily during 14 and 21 days caused statistically significant rise in the number of the Gomori-positive glial cells around the third cerebral ventricle of mice. In addition, the nuclei of the Gomori-positive glial cells markedly increased in volume.