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Biomedical subjects

D Zicha

Publications and source records attributed to D Zicha.

28 records · Page 2Linked to original sources

Origins of the parasitophorous vacuole membrane of the malaria parasite: surface area of the parasitized red cell.

There is conflicting evidence on whether the parasitophorous vacuole membrane, in which the malaria parasite becomes encapsulated when it enters the red cell, represents a part of the host cell membrane or is derived, at least in part, from the parasite. We have measured the surface area of populations of red cells before and after invasion by up to four merozoites of the malaria parasite, Plasmodium falciparum. The dimensions of the merozoite are such that, if it enveloped itself entirely in host cell membrane during entry, the loss of surface area would amount to some 4 square microns 2 or 3% of the total for each parasite internalized. Our measurements show that within the 99% level of confidence any loss of surface area is less than 1 square micron 2 per parasite internalized. Area measurements on red cells that have been allowed to lose known proportions of their membrane by metabolically induced vesiculation reveal, moreover, that diminutions in surface area in the range of interest are readily detectable. Our observations on recently invaded (young ring-stage) parasites appear to exclude any significant change in surface area of the host cell following invasion. This implies that, if indeed there is internalization of host cell membrane lipid on invasion, as the best evidence shows, it is compensated by parasite-derived lipid, and conversely the parasitophorous vacuole membrane probably contains a contribution of parasite-derived material, presumably that seen to be discharged by the apical organelles, the rhoptries, at the time of invasion.

Animals↗

Actin cytoskeleton and motility in rat sarcoma cell populations with different metastatic potential.

We have studied the organisation of the actin cytoskeleton in three related rat sarcoma cell populations of differing malignancy. They were derived by neoplastic progression from a population which had transformed spontaneously in vitro, and were distinguished by their ability to give rise to reproducibly different numbers of metastases, ranging from 10% to 80% of the animals inoculated. We found characteristic differences in the arrangement of the actin cytoskeleton. Confocal three-dimensional microscopy showed that nearly all of the least malignant population contained conspicuous actin stress fibres lying in the lower part of the cell parallel to the substratum and no other actin structures. Actin in the intermediate population was typically situated in a diffuse layer underlying the whole plasma membrane, in which no fibres could be seen. Two thirds of the most malignant population consisted of more rounded cells filled with a three-dimensional network of fine oblique actin fibres. There were focal contacts in all these cells; their area showed a regular decrease from 1.3 microns 2 to 0.4 microns 2. The differences in actin distribution were accompanied by differences in motility, which increased as malignancy increased. When individual cells were fixed after they had been tracked by time-lapse, their cytoskeleton type correlated with the speed at which they had moved. All these differences were enhanced at low pH. These findings point to the possibility that the three-dimensional network of fine actin fibres in acid culture could be a measure of the malignant potential of transformed cells in vitro.

Actins↗

Phase-shifting interference microscopy applied to the analysis of cell behaviour.

The theory of phase-shifting interferometry is not new but it is only recently, with the advent of solid-state detector arrays and fast image processors, that it has become a practical imaging technique. In conjunction with transmission interference microscopy, phase-shifting presents a new way of introducing contrast into the images of transparent microscopic objects such as cultured cells. An earlier paper from our laboratory has emphasised the advantages of transmission interference microscopy over phase contrast or differential interference contrast microscopy for the computerised analysis of cell behaviour. Phase-shifting greatly improves the accuracy, long-term stability and range of application of this technique but it has not previously, to our knowledge, been combined with transmission interference microscopy for the study of cultured cells. The resulting image is especially well suited to quantitative analysis by computer since it is a direct representation of the distribution of non-aqueous cellular material in the specimen. The image is not degraded by uneven illumination; by heterogeneous sensitivity of the detector array; or by differential absorption of light in the optics or specimen. Our main purpose in developing the method is to obtain sequences of images of the motile behaviour of cells in culture for analysis by computer. This type of analysis is potentially a powerful tool for studying the motile responses of cells and the operation and control of their locomotory machinery. Not only can the method be used for studying cell translocation and the dynamics of intracellular movement of non-aqueous material, but it is now possible to study in detail the time course of growth in individual cultured cells.

Animals↗

Long-term chemotaxis of neutrophils in stable gradients: preliminary evidence of periodic behavior.

We observed and recorded the behavior of human neutrophils in stable linear gradients of the chemotactic attractant f-Met-Leu-Phe in a recently developed, direct-viewing, chemotaxis chamber. The cells were kept cold for the first hour after setting up the chemotaxis chamber in order to establish a stable gradient before allowing the cells to spread and begin to move by warming them up to 37 degrees C. We found that the cells showed very significant chemotaxis at different times throughout the 2-hour period of observation but, unexpectedly, the activity occurred in periodic or quasi-periodic cycles during which the cells entered phases of undirected motility lasting for several minutes. We discuss whether the periodicity is a population or single cell-based phenomenon and briefly speculate on its possible analogy to the well-known periodic chemotaxis of slime mould amoebas.

Chemotaxis, Leukocyte↗

A new direct-viewing chemotaxis chamber.

A new form of chamber for studying chemotaxis, similar in principle to the Zigmond chamber, allows the behaviour of the cells in a linear concentration gradient to be observed directly. The chamber was developed mainly for studying chemotaxis in fibroblasts using interferometric microscopy and the main design criteria were that it should have better optical characteristics, a higher dimensional precision and better long-term stability than the Zigmond chamber. It is made entirely from glass by grinding a blind circular well centrally in the counting platform of a Helber bacteria counting chamber. This procedure leaves an annular 'bridge', approximately 1 mm wide, between the new inner circular well and the original outer annular well. This bridge fulfils the same function as the linear bridge of the Zigmond chamber but the precise construction of the counting chamber ensures that a gap of 20 microns between bridge and coverslip can be accurately and repeatedly achieved when the chamber is assembled. It is envisaged that the improved optical clarity, dimensional accuracy and long-term stability of the new chamber will be advantageous in other applications, particularly in studies requiring critical microscopy or a precise knowledge of the gradient and in studies of cells, such as fibroblasts, that move much more slowly than neutrophils.

Chemotaxis↗

Quasi-dynamic test of in vitro cell migration developed to characterize dividing cells of spontaneously metastasizing rat sarcomas.

A "Quasi-dynamic Test of in vitro Cell Migration" (QTCM) was developed in which the quantitative estimation of migration of dividing cells is based on a comparative evaluation of their dispersion in single-cell-derived colonies after 72 h of clonal growth under standard and test conditions. Expert visual evaluation of microscopic images of cell colonies including estimation of dispersion of cells in the colonies is checked and made more precise by computerized evaluation of digitized outlines of cells in the colonies. Using QTCM, distinctions in the pattern of migration between three neoplastic cell populations differing by their origin and degree of malignancy were found. Although transfer of cells into slightly acid conditions did not always increase the mean value of cell dispersion in the colonies, each cell population was found to include a cell fraction that did manifest considerably greater migration and this fraction was markedly larger in the more malignant cell populations. The results of both types of evaluation, visual and computerized, were in mutual accord, with accuracy being greater on the computer side. Thus, the QTCM proved useful for qualified estimation and/or computer measurement of in vitro migration of dividing cells on a population level.

Analog-Digital Conversion↗

Cathepsin B in cells of two rat sarcomas with different rates of spontaneous metastasis.

The cysteine proteinase cathepsin B (EC 3.4.22.1) is believed to take part in biochemical processes underlying tumor metastasis. In the present study, the cellular localization, intracellular levels and extracellular release of cathepsin B activity were examined in vitro in cells of two rat sarcoma variants, LW13K2 and RPS, differing in their capacity to metastasize spontaneously to the lung of syngeneic LEW/CUB rats. The LW13K2 sarcoma metastasizes rarely, whereas the LW13K2-derived RPS variant produces a metastasis incidence of above 50%. Using fluorescent cytochemical staining, microgranular reaction centers of cathepsin B were observed in the cell cytoplasm, in some cellular processes, with apical localization in some of them, as well as at the extreme cell periphery in cells of both sarcoma variants. The appearance of this distribution of cathepsin B activity was delayed in the RPS variant. Biochemically, the intracellular level of cathepsin B activity was significantly higher in homogenates of LW13K2 cells than RPS cells. In contrast to the intracellular enzyme activity, RPS cells cultured in serum-free medium at pH 6.5 released a substantially higher amount of cathepsin B activity than LW13K2 cells into the extracellular environment; at pH 7.4 the initially higher release of cathepsin B activity from RPS cells later equalized with that from LW13K2 cells. Taken together, the results indicate that changes of pericellular pH can modulate the extracellular release of cathepsin B in both sarcoma cell variants and suggest that the rate of cathepsin B release under conditions of mildly acid pericellular pH could be related to the incidence of metastases observed in these rat sarcoma variants. Total intracellular cathepsin B activity did not exhibit positive correlation with the metastatic potential of the studied rat sarcoma variants.

Animals↗

[Use of a computer program in the diagnosis of brain tumors].

The FEL-EXPERT was used to improve the diagnosis of brain tumors. Basic structure and decision process of the system were characterized. In a group of 70 testing cases the correct diagnosis was established at the 1st possibility in 83%, as the 1st or 2nd possibility in 91% and as the 1st or 2nd or 3rd possibility in 17%.

Artificial Intelligence↗