Trypanosoma brucei: biochemical and morphological studies of cytotoxicity caused by normal human serum

The biochemical and morphological events which accompany lysis of Trypanosoma brucei by normal human serum have been described. The prelytic events include loss of infectivity and rapid cation shifts across the cell membrane. This is followed by cell swelling, fraying of the surface coat of the cell, loss of intracellular organelles, and eventually cell lysis. The data presented are consistent with a colloid osmotic mechanism of lysis induced by irreversible acute damage to the normal permeability properties of the trypanosome plasma membrane.     

The solubilization and morphological change of human platelets in various detergents

The solubilization of human gel-filtered platelets by octyl glucoside, Triton X-100, dodecylsulfate, and deoxycholate was compared from the analysis of (1) cell lysis, (2) marker leakiness, and (3) component solubility. These analyses all revealed that the effect of detergent concentration on the solubilization of platelets by these detergents was exerted in three stages, i.e., the prelytic, lytic, and complete platelet-lysis stages. These analyses also indicated several differences among platelets in these detergents. (i) The ratio of the platelet-saturation concentration (PSC) to critical micellar concentration (CMC) was about 1/2 for octyl glucoside. Triton X-100 and dodecylsulfate, while it was close to 1 for deoxycholate. (ii) Platelets in octyl glucoside. Triton X-100, and dodecylsulfate all showed parallel curves in cell lysis, protein solubilization and marker leakiness, while the platelet lysis in deoxycholate was identical to the phospholipid solubilization. (iii) The solubility curves of various components in Triton X-100 and deoxycholate were parallel. However, the solubility of cholesterol in octyl glucoside was lower than that of protein and phospholipid. In dodecylsulfate, the solubility of phospholipid and cholesterol was very low in comparison with that of protein. In addition, morphological studies using scanning electron microscopy (scanning EM) revealed that the solubilization by octyl glucoside or Triton X-100 might occur via membrane area expansion. On the other hand, the solubilization by dodecylsulfate or deoxycholate showed membrane vesiculation prior to cell lysis. Moreover, in the prelytic stage, the morphological change in platelets in octyl glucoside showed only concentration dependence by swelling to an ellipsoid and then to a sphere. However, the morphological change in platelets in the other three detergents was dependent not only on the detergent concentration but also on prolonged incubation. Specifically, in Triton X-100, the cells initially changed to spiculate discs and then reached their final shape as swollen discs with surface invagination. In dodecylsulfate and deoxycholate the morphological changes were almost the same. The cell initially deformed in shape to a spiculate disc and finally to a stretched-out flat form. The results are discussed according to the bilayer couple hypothesis. Also, in the prelytic stage, these detergents caused inhibition of the response of platelets to collagen and ADP-fibrinogen.     

Cell Lysis Due to Ultrasound Gel In Fine Needle Aspirates; an Important New Artefact In Cytology

An important new artefact in cytopathology is described. the initial observation of the artefact followed contamination of a breast fine needle aspiration (FNA) sample by ultrasound gel which was used to localize the lesion. the changes proved reproducible ex vivo. the changes varied depending on the conditions and degree of contamination, and ranged from cell swelling to leakage of nuclear chromatin and cell lysis. This artefact is discussed in the context of other major sources of cytology artefact. Pathologists and radiologists should beware of the detrimental effects of ultrasound gel on cytology specimens.     

Adaptive reactions of mycoplasmas in vitro: "viable but unculturable forms" and nanocells of Acholeplasma laidlawii

The adaptation of Acholeplasma laidlawii to conditions unfavorable for growth has been found to be accompanied by cell transformation into special morphological structures known as ultramicroforms (nanocells). The ratio of the cells of the two morphological types in the population depended on the growth conditions. Nanocells retained viability for a long time under conditions unfavorable for growth and showed resistance to stressors. Reduction in the cell size occurred due to unequal division, which involved the loss of cytoplasmic material. A. laidlawii ultramicroforms (nanocells) were able to restore proliferative activity and to revert to their initial vegetative form; they measured less than 0.2 microm and are the smallest cells known at present. Nanocells formed in vitro under exposure to abiogenic stressors may correspond to the A. laidlawii minibodies observed in infected plants upon exposure to biogenic stressors. The transformation of A. laidlawii cells into ultramicroforms was accompanied by condensation of the nucleoid, a change in the polypeptide spectrum, and a change in the availability of rRNA operons for in vitro amplification. All these changes are indicative of reorganization of the genetic and metabolic systems of mycoplasmas.     

Energetic swelling and lysis of mitochondria

B1 avitaminosis presents a typical example of energetic mitochondrial degeneration. The alterations--swelling and lysis of mitochondria--are accompanied by dilatation with formation of megamitochondria, by appearance of blebs and tearings of the mitochondrial membranes, by progressive fading of the matrix, by swelling of the mitochondrial membranes with lysis of the cristae advancing from centre towards periphery, by vacuolar transformation of the cell and by development of onionoid bodies (corpora cepiformia).     

Human serum lyses Trypanosoma brucei by triggering uncontrolled swelling of the parasite lysosome

Trypanosoma brucei brucei infects a wide range of mammals, but is unable to infect humans because this subspecies is lysed by normal human serum (NHS). The phenotype of cellular lysis is debated. For some authors the lysosome undergoes osmotic swelling due to massive influx of chloride ions from the cytoplasmic compartment, but others describe multiple small cytoplasmic vacuoles and general swelling of the cellular body. Using population-level imaging of live immobilized trypanosomes throughout the lysis process, we report that specific swelling of the lysosome is a genuine and major characteristic of NHS-mediated lysis and that this phenotype is independent of the strain of trypanosomes and of NHS aging or damaging. Thus, irrespective of experimental conditions NHS reproducibly induced the swelling of the parasite lysosome.     

Morphologic changes in cultures of different tissues exposed to the toxins of C1. perfringens types B, C, E and F]

There were revealed morphological peculiarities of the action of C1. perfringens toxins, types B, C, D, E and F on the cultures of fibroblasts of chick embryo, amniotic cells and intestinal tissue. The toxin type B was characterized by a marked vocuolization of the cell cytoplasm; the action of the toxin of type C was expressed in the swelling of the nuclei and the lysis of the chromatine substance, the toxin of type E casued kariorhexis, and the toxin of type F--hyperchromatosis of the nuclei. All the cultures proved to be insensitive to the toxin of type D. Peculiarity of the morphological affection of the cells permitted to differentiate toxin of type B in the cultures of the fibroblasts of chick embryo, whereas the toxins of types C, E and F--in the cultures of the amniotic cells under control of the reaction of neutralization with the homologous antitoxic sera.     

Biological activities and pore formation of Clostridium perfringens beta toxin in HL 60 cells

Clostridium perfringens beta toxin is an important agent of necrotic enteritis. Of the 10 cell lines tested, only the HL 60 cell line was susceptible to beta toxin. The toxin induced swelling and lysis of the cell. Treatment of the cells with the toxin resulted in K+ efflux from the cells and Ca2+, Na+, and Cl- influxes. These events reached a maximum just before the cells were lysed by the toxin. Incubation of the cells with the toxin showed the formation of toxin complexes of about 191 and 228 kDa, which were localized in the domains that fulfilled the criteria of lipid rafts. The complex of 228 kDa was observed until 30 min after incubation, and only the complex of 191 kDa was remained after 60 min. Treatment of the cells with methyl-beta-cyclodextrin or cholesterol oxidase blocked binding of the toxin to the rafts and the toxin-induced K+ efflux and swelling. The toxin-induced Ca2+ influx and morphological changes were inhibited by an increase in the hydrodynamic diameter of polyethylene glycols from 200 to 400 and markedly or completely inhibited by polyethylene glycol 600 and 1000. However, these polyethylene glycols had no effect on the toxin-induced K+ efflux. The toxin induced carboxyfluorescein release from phosphatidyl-choline-cholesterol liposomes containing carboxyfluorescein and formed an oligomer with 228 kDa in a dose-dependent manner but did not form an oligomer with the 191-kDa complex. We conclude that the toxin acts on HL 60 cells by binding to lipid rafts and forming a functional oligomer with 228 kDa.     

THE CYTOTOXIC PRINCIPLE OF THE PHYTOFLAGELLATE PRYMNESIUM PARVUM

The cytotoxic events leading to lysis induced in Ehrlich ascites tumor (E.A.) cells by Prymnesium parvum cell extracts were followed microscopically and measured quantitatively as changes in E.A. cell volume, uptake of trypan blue, and release of macromolecular constituents from the cells. Cell swelling was the most immediate response to P. parvum cytotoxin, while cell death and lysis were later events distinguished by a decline in cell volume, uptake of dye, and appearance of cellular macromolecules free in the incubation medium. The pH and temperature were shown to affect the outcome of the lytic sequence. At either low pH or temperature, cells swelled but did not lyse until the pH or temperature was raised. On the other hand, cells swollen at the higher pH or temperature could be protected from lysis by lowering either the pH or the temperature.     

Plaque assay of avian sarcoma viruses using casein.

The caseinolytic activity of several strains of Rous sarcoma virus (RSV), conditional and nonconditional mutants of RSV, and nontransforming avian leukosis viruses was investigated. Only those viruses capable of transforming chick fibroblasts in vitro induced lysis of casein incorporated into an agar overlay. Lysis produced distinct clear areas in the turbid casein-agar gel which allowed a quantitative "plaque" assay of cell transformation. Casein plaque formation could not be separated from morphological conversion in cultures infected by wild-type RSV strains. In plates infected by mutants temperature sensitive for transformation, the caseinolytic activity appeared to be affected by temperature to a lesser extent than morphological conversion.     

Effect of sodium hydroxybutyrate on the ultrastructure of cross-striated muscle tissue myocytes during physical exercise

The ultrastructure of myocytes of the rat myocardium and skeletal muscles was studied in the control during physical exercise and under conditions of two-week sodium hydroxybutyrate pretreatment. It was shown that single maximum physical exercise caused significant changes in the fine structure of cardiomyocytes and somewhat less changes in a pronounced intermyofibrillar edema, the swelling of mitochondria and an acute fall of the glycogen level. A two-week sodium hydroxybutyrate pretreatment prevented the changes in the myocytes. The observed structure normalization induced by the drug is likely to be due to the specific nature of its metabolic transformation and to the effect on the energy exchange.     

Characterization of Lysis of the Multicellular Cyanobacterium Limnothrix/Pseudanabaena sp. Strain ABRG5-3

The cyanobacterium semi-filamentous multicellular strain ABRG5-3 undergoes cell lysis as a unique feature that occurs due to growth condition changes from normal cultivation with shaking to static cultivation without shaking in liquid culture (Nishizawa et al., 2010). Microscopic observation and energy dispersive X-ray spectrometer (EDX) analysis have revealed that lysis is involved in the accumulation of polyphosphate compounds and the disintegration of thylakoid membranes in cells. Static cultivation, dark or red light exposure, and temperature (22 to 42 °C) conditions were found to be effective factors for the induction of lysis. Moreover, stress induced by salts, osmotic pressure with sucrose, and the depletion of nitrogen or phosphate in cultures also induced ABRG5-3 cell lysis. Based on these results, we discuss lysis and its utilization in the biotechnology industry.     

Development of Coliphage N4: Ultrastructural Studies

The basic properties of bacteriophage N4 development have been investigated in Escherichia coli Hfr 3300 under one-step growth and high cell density conditions. N4r(+) -infected bacteria are lysis inhibited in mass culture, burst asynchronously starting 180 min postinfection, and release over 3,000 phage per cell. During lysis inhibition the bacteria continuously elongate, increase in girth, and undergo characteristic morphological changes represented by the appearance of dark spots located at the cell poles. In thin sections, during the late stages of replication and assembly, the phage particles are localized exclusively in restricted areas of the cytoplasm near the polar regions. Large paracrystalline arrays of virions are found in over 7% of the cells before lysis. The most common mechanism of lysis consists in the formation of bulges located at random in the cell circumference; these burst and, without extensive disruption of the cell wall, the phage progeny escapes into the medium.     

Mechanics and Dynamics of Bacterial Cell Lysis

Membrane lysis, or rupture, is a cell death pathway in bacteria frequently caused by cell wall-targeting antibiotics. Although previous studies have clarified the biochemical mechanisms of antibiotic action, a physical understanding of the processes leading to lysis remains lacking. Here, we analyze the dynamics of membrane bulging and lysis in Escherichia coli, in which the formation of an initial, partially subtended spherical bulge (“bulging”) after cell wall digestion occurs on a characteristic timescale of 1 s and the growth of the bulge (“swelling”) occurs on a slower characteristic timescale of 100 s. We show that bulging can be energetically favorable due to the relaxation of the entropic and stretching energies of the inner membrane, cell wall, and outer membrane and that the experimentally observed timescales are consistent with model predictions. We then show that swelling is mediated by the enlargement of wall defects, after which cell lysis is consistent with both the inner and outer membranes exceeding characteristic estimates of the yield areal strains of biological membranes. These results contrast biological membrane physics and the physics of thin, rigid shells. They also have implications for cellular morphogenesis and antibiotic discovery across different species of bacteria.     

大黄鱼源溶藻弧菌的鉴定及其菌蜕制备

【背景】菌蜕是诱导Phi X174噬菌体裂解基因E(Lysis E)在革兰氏阴性菌中表达后所获得无细胞内容物的细菌空壳。菌蜕生物安全性高,能以类似活菌方式诱导机体产生良好的系统和黏膜免疫应答。【目的】对分离自患溃疡病大黄鱼肝脏中的病原菌株16-3进行种属鉴定,利用温控调节表达系统控制Phi X174噬菌体裂解基因E在该菌株中的表达来制备菌蜕,为防控鱼类溶藻弧菌感染提供有效手段。【方法】采用形态特征观察、生理生化特性测定及16S r RNA基因序列分析等方法对菌株16-3进行鉴定;构建温控裂解质粒p BV220-Lysis E,并将其电转至溶藻弧菌菌株16-3,形成重组溶藻弧菌菌株16-3(p BV220-Lysis E);将不同起始浓度的重组溶藻弧菌培养物同时进行42°C升温诱导,比较其溶菌动力曲线和裂解效率的差异;在最佳条件下制备溶藻弧菌菌株16-3菌蜕,电镜观察其形态与结构,采用倾注平板法测定冻干菌蜕中的活菌数。【结果】综合菌株16-3在形态、生理生化及16S r RNA基因系统发育等方面的特性,确定其为溶藻弧菌;构建了温控裂解质粒p BV220-Lysis E和重组溶藻弧菌菌株16-3(p BV220-Lysis E);溶藻弧菌菌株16-3菌蜕制备的最佳条件是选择起始浓度OD600为0.3的菌液进行诱导,诱导3 h后即可收获菌蜕,其裂解效率为96.9%,但经冻干处理后的菌蜕无活菌残留;电镜观察发现菌株16-3菌蜕保持原细胞的基本形态,但细胞表面有明显的溶菌孔道,且由于细胞内容流失而使细胞表面发生皱缩。【结论】制备出溶藻弧菌菌株16-3菌蜕,为其作为疫苗或疫苗递送载体奠定了基础。     

Antibacterial activities of beta-lactamase inhibitors associated with morphological changes of cell wall in Helicobacter pylori

Background. Recent study has demonstrated that β‐lactamase inhibitors including clavulanate, sulbactam and tazobactam have an vitro antibacterial effect on Helicobacter pylori. Here we describe the relationship between viability and cell profiles of H. pylori exposed to β‐lactamase inhibitors and some antibiotics in a short‐time course. Materials and methods. The antibacterial effects of β‐lactamase inhibitors including clavulanate, sulbactam and tazobactam on the bacterial viability of and morphological changes in H. pylori ATCC43504 were examined. Results. The β‐lactamase inhibitors such as clavulanate and sulbactam alone decreased the viable counts of H. pylori, depending on the antibiotic concentrations. Exposure to these β‐lactamase inhibitors resulted in morphological changes of cell shape, cell‐wall disintegration and cell lysis. Among these β‐lactamase inhibitors, clavulanate was the most active, causing a decrease in viable counts and morphological changes such as short filamentous to sphaeroplast formation and lysis. One × minimum inhibitory concentration (MIC) of amoxicillin plus 1 × MIC of clavulanate decreased viable counts effectively compared with 1 × MIC of amoxicillin or 1 × MIC of clavulanate alone, and induced morphological changes of cell shape and cell wall. Conclusion. Our results suggest that the β‐lactamase inhibitors alone have concentration‐dependent antibacterial activities against H. pylori and affect the morphology of the cell shape and the cell wall in vitro.     

An interlaboratory comparison of enhanced morphological transformation of Syrian hamster embryo cells cultured under conditions of reduced bicarbonate concentration and pH

Initial studies performed in our laboratory indicated that early passage Syrian hamster embryo (SHE) cells exhibit optimal clonal proliferation when cultured in medium with a sodium bicarbonate concentration of 8.9 mM and pH of 6.70 instead of 44 mM and pH 7.35 as used previously by others. Subsequent studies indicated that morphological transformation frequency induced by benzo[a]pyrene (BP) was also enhanced at pH 6.70 compared to 7.35 and the level of enhancement was affected by cell density and duration of culture. With optimal conditions identified, the carcinogens BP, 3-methylcholanthrene, N-methyl-N'-nitro-N-nitrosoguanidine, 2-acetylaminofluorene and the non-carcinogen anthracene were tested at pH 6.70 and 7.35 in our laboratory and at Microbiological Assoc. Inc. under code. Additionally, the non-carcinogens 4-acetylaminofluorene, and caprolactam were tested in our laboratory. Results from these studies indicate that all carcinogens tested caused a significant increase in morphological transformation frequency compared to controls at pH 6.70. In contrast, only BP caused a significant increase in the morphological transformation frequency at pH 7.35. The non-carcinogens did not significantly increase the morphological transformation frequency compared to controls. Interlaboratory comparisons were in qualitative agreement despite the fact that different lots of serum and hamster cell isolates were used by the two laboratories. However, different dose-response curves for the various chemicals were observed between the two labs. It was also demonstrated that the enhanced morphological transformation frequency is not due to a decrease in culture medium osmolality or Na concentration, a condition which accompanies media with a reduced bicarbonate concentration and pH. These results demonstrate that the chemicals tested, low pH transformation of SHE cells agrees with carcinogenic potential and that assay variability is minimized. The implications of these results regarding use of the SHE cell assay as a short-term test for predicting the carcinogenic potential of chemicals are discussed.     

Adaptive Reactions of Mycoplasmas In Vitro: “Viable but Unculturable Forms” and Nanocells of <Emphasis Type="Italic">Acholeplasma laidlawii</Emphasis>

The adaptation of Acholeplasma laidlawii to conditions unfavorable for growth has been found to be accompanied by cell transformation into special morphological structures known as ultramicroforms (nanocells). The ratio of the cells of the two morphological types in the population depended on the growth conditions. Nanocells retained viability for a long time under conditions unfavorable for growth and showed resistance to stressors. Reduction in the cell size occurred due to unequal division, which involved the loss of cytoplasmic material. A. laidlawii ultramicroforms (nanocells) were able to restore proliferative activity and to revert to their initial vegetative form; they measured less than 0.2 µm and are the smallest cells known at present. Nanocells formed in vitro under exposure to abiogenic stressors may correspond to the A. laidlawii minibodies observed in infected plants upon exposure to biogenic stressors. The transformation of A. laidlawii cells into ultramicroforms was accompanied by condensation of the nucleoid, a change in the polypeptide spectrum, and a change in the availability of rRNA operons for in vitro amplification. All these changes are indicative of reorganization of the genetic and metabolic systems of mycoplasmas.__________Translated from Mikrobiologiya, Vol. 74, No. 4, 2005, pp. 498–504.Original Russian Text Copyright © 2005 by Chernov, Mukhametshina, Gogolev, Abdrakhimov, Chernova.     

Structural changes of mitochondria related to apoptosis

The original concept of apoptosis stressed the morphological changes of the nucleus, condensation with the aggregation of chromatin, and the intactness of intracellular organelles including mitochondria. However, the application of molecular biology and flow-cytometric techniques to the research field of apoptosis has led to the proposal of the apoptotic processes which emphasizes the 'swelling of mitochondria' due to the opening of the 'permeability transition pores' of the mitochondrial membranes followed by a series of events including the collapse of the transmembrane potential of mitochondria and release of cytochrome c from mitochondria into the cytosol. Enlargement of mitochondria induced by various pathological conditions are classified into two categories: the swelling and the formation of megamitochondria (MG). Recently, we have found that free radical-induced formation of MG is succeeded by apoptotic changes of the cell. If the MG formation is actually related to apoptosis, this will be a new aspect of the structural changes of mitochondria involved in apoptosis besides the simple swelling of mitochondria. First, we will discuss the 'swelling of mitochondria' which characterizes the currently accepted hypothesis on the apoptotic processes of the cell, as described above, in the light of the literature. Second, the mechanisms controlling the size and distribution of mitochondria in the cell are dealt with paying special attention to the genetic regulation and cytoskeletons. Third, we have tried to characterize the MG formation to correlate apoptotic changes of the cell. Finally, we will discuss several problems to be solved in the future which involve mitochondria in apoptotic processes of the cell.     

Morphogenic effect of 2-deoxy-D-arabino-hexose onRhodosporidium toruloides

The presence of 2-deoxy-D-arabino-hexose in the growth medium caused marked morphological changes in the cells of Rhodosporidium toruloides. The originally elongated ellipsoidal cells grew spherically in the presence of the deoxy-sugar, displayed differences in cell division and separation, and were larger than the control cells. After exhaustion of glucose from the medium the cells died, although no lysis was observed. The morphological changes were accompanied by significant alterations in the carbohydrate composition of the cell wall. The wall of R. toruloides grown in the presence of the deoxy-sugar contains higher proportions of chitin and glucan, while the relative contents of mannose and galactose polymers decreased drastically in comparison to normal cells.