Prediction of cervical neoplasia diagnosis groups. Discriminant analysis on digitized cell images

The purpose of this study was to develop discriminant analysis models for predicting cervical dysplasia/neoplasia case diagnoses using cytometric features derived from the digital image analysis of cell monolayers. The data base consisted of 925 cells from 27 cases diagnosed either as moderate dysplasia (n = 10), severe dysplasia (n = 5), carcinoma in situ (n = 8) or invasive carcinoma (n = 4) on both tissue biopsy and monolayer preparations. Cell features examined were cell diameter, nuclear diameter, nuclear mean optical density (OD), nuclear integrated OD (IOD), nuclear OD standard deviation, normalized IOD, nuclear texture and nuclear-cytoplasmic ratio. Features derived from cells visually classified as moderate dysplasia correctly predicted the case diagnosis of moderate dysplasia versus more severe disease for 85% of the cells. Prediction models using summary measures (mean and variance) derived from all visually classified abnormal cells within each case correctly separated all cases into their respective diagnostic categories. These findings suggest that dysplastic cells in a cytologic sample have features that collectively reflect the tissue diagnosis, regardless of the visual differences among the cells. Such information has potential use for diagnosis and possibly for prognosis.     

A program system for interactive measurements on digitized cell images.

Using a high precision image scanner and a PDP-8/F minicomputer, we have developed a program system for interactive measurements on microscopic images. By giving simple keyboard commands, the operator can run the image scanner and manipulate the digitized images. The interface between the operator and the microscope-computer system is a Tektronix 4010 graphic terminal. The system allows objects to be isolated and parameters to be calculated from each object, e.g., parameters characterizing shape of the object, irregularity in light transmission over the object, area, integrated light transmission, etc. Objects are isolated and parameters are calculated under complete operator control using interactive computer graphics technique. Calculated parameters may be stored in dedicated data records, which are stored in files for later statistical analysis. The system also includes a statistical evaluation part. Technically, the system consists of a command scanner, which translates commands into internal representation, a parser, which checks the syntax of the commands, and an interpreter, which executes the commands. The system is designed so that new commands can be added easily.     

Transblot and cytochemical identification of avidin-interacting proteins in mitochondria of cultured cells

Cell lysates prepared from 3T3-L1 cells were analyzed by western blotting using the avidin-biotin complex system and anti-Bax antibody. The antibody interacted with bands of proteins with estimated molecular weights of 120, 74, 72, and 25 kDa. However, only the 25-kDa band was detected with the anti-Bax antibody when the direct immunoblotting method was used. Peroxidase-conjugated avidin interacted with the 120-, 74-, and 72-kDa bands. This interaction was not limited to 3T3-L1 cells, because peroxidase-avidin also interacted with these three proteins in MC3T3-E1, YROS, Saos-2, MG63, SCCKN, and SCCTF cells although the staining intensity was different in each cell type. Avidin-peroxidase also interacted with these three proteins in the mitochondria-containing fractions prepared from 3T3-L1 cells. FITC-streptavidin was also localized in mitochondria in the cultured cells. The localization of avidin/streptavidin-interacting proteins in mitochondria was confirmed by using double staining with FITC-streptavidin and Mito-tracker.     

Altered distribution of mitochondria and actin fibers in 3T3 cells cultured on microcarriers.

The mitochondria and actin fibers of 3T3 fibroblasts cultured on microcarriers in spinner flasks were visualized using fluorescent stains. In contrast to cells grown on planar surfaces under static or steady laminar flow conditions, cells exposed to higher levels of turbulent agitation do not form actin stress fibers. Greater agitation also leads to a more diffuse appearance of the mitochondria and a wider distribution of them throughout the cytoplasm. This response may indicate damaged mitochondria, as similar results have been reported for chemical toxins.     

Interaction between neurofilaments and mitochondria in cultured cells of the rat hippocampus

Interaction between neurofilaments and mitochondria was studied on the model of cultured hippocampal cells of newborn rats. A treatment by specific toxins -iminodipropionitril or hexanedione resulted in a disintegration and translocation of neurofilaments in the cultured neurons. These effects were accompanied by a considerable decrease in dimensions of mitochondria, an increase in their elongation coefficient, a noticeable increase in spatial density of these organelles, and their translocation within the perinuclear layer of cytoplasm. The role of neurofilaments in the intraneuronal distribution of mitochondria and modifications of their functional state is discussed. The neurofilament system is supposed to considerably influence the processes of division, growth, and translocation of the mitochondria.Neirofiziologiya/Neurophysiology, Vol. 27, No. 1, pp. 3–10, January–February, 1995.     

Isolation of biogenetically competent mitochondria from mammalian tissues and cultured cells

This article describes a quick basic method adapted for the purification of mammalian mitochondria from different sources. The organelles obtained using this protocol are suitable for the investigation of biogenetic activities such as enzyme activity, mtDNA, mtRNA, mitochondrial protein synthesis, and mitochondrial tRNA aminoacylation. In addition, these mitochondria are capable of efficient protein import and the investigation of mtDNA/protein interactions by DNA footprinting is also possible.     

The identification of calmodulin-binding sites on mitochondria in cultured 3T3 cells

We have uniformly labeled calmodulin with tetramethyl rhodamine isothiocyanate (CaM-RITC) and used the derivative as a molecular probe in order to identify available, unoccupied calmodulin-binding sites. In mildly fixed (3% formalin) cultured 3T3 cells, the biologically active CaM-RITC bound predominantly to mitochondria. Binding was markedly reduced in the presence of 1 mM EGTA. Stelazine, a phenothiozine which binds to calmodulin, prevented the interaction of CaM-RITC with mitochondrial sites. A 10 fold excess of unlabeled CaM competitively inhibited binding. Fluorescently labeled troponin C and parvalbumin did not bind to mitochondria on any other cellular organelle. Rhodamine (TMRITC) alone did not bind to 3T3 mitochondria. Similar results were obtained using ¹²⁵I-calmodulin binding to isolated rat liver mitochondria. When solubilized mitochondrial proteins were subjected to calmodulin-Sepharose affinity chromatography and eluted with 1 mM EGTA, there were two major polypeptides 120,000 and 67,000 daltons and at least three minor species (100,000, 60,000 and 40,000 daltons). The interaction required an active Ca²⁺-CaM complex and is specific for CaM. Double fluorescent staining with CaM-RITC and fluorescein-labeled antibodies to tubulin and DNAase I revealed a mitochondrial distribution pattern similar to that of microtubule arrays but unrelated to actin cabling. There was no evidence that CaM-RITC directly interacted with either microtubules or microfilaments.     

Measuring wood consumption by subterranean termites (Isoptera: Rhinotermitidae) with digitized images

A method is described in which consumption by termites of a feeding block composed of 36 thin (2-mm) wooden slats is estimated with a video image analysis. The significant regression between dry wood weight loss and decreased pixel count of digitized images demonstrates that the video image technique may be a useful tool to quantify termite feeding. Time required to measure wood consumption with the video image technique (approximately 11.5 min per sample) was significantly less than that for the manual method of washing, and weighing wooden blocks damaged by termite feeding (approximately 21.0 min per sample).     

Replication of mitochondrial DNA occurs throughout the mitochondria of cultured human cells

Replication of mitochondrial DNA (mtDNA) is dependent on nuclear-encoded factors. It has been proposed that this reliance may exert spatial restrictions on the sites of mtDNA replication within the cytoplasm, as a previous study only detected mtDNA synthesis in perinuclear mitochondria. We have studied mtDNA replication in situ in a variety of human cell cultures labeled with 5-bromo-2'-deoxyuridine. In contrast to what has been reported, mtDNA synthesis was detected at multiple sites throughout the mitochondrial network following short pulses with bromodeoxyuridine. Although no bromodeoxyuridine incorporation was observed in anuclear platelets, incorporation into mtDNA of fibroblasts that had been enucleated 2 h prior to labeling was readily detectable. Blotting experiments indicated that the bromodeoxyuridine incorporation into mtDNA observed in situ represents replication of the entire mtDNA molecule. The studies also showed that replication of mtDNA occurred at any stage of the cell cycle in proliferating cells and continued in postmitotic cells, although at a lower level. These results demonstrate that mtDNA replication is not restricted to mitochondria in the proximity of the nucleus and imply that all components of the replication machinery are available at sufficient levels throughout the mitochondrial network to permit mtDNA replication throughout the cytoplasm.     

Motor deficits in schizophrenia quantified by nonlinear analysis of postural sway

Motor dysfunction is a consistently reported but understudied aspect of schizophrenia. Postural sway area was examined in individuals with schizophrenia under four conditions with different amounts of visual and proprioceptive feedback: eyes open or closed and feet together or shoulder width apart. The nonlinear complexity of postural sway was assessed by detrended fluctuation analysis (DFA). The schizophrenia group (n = 27) exhibited greater sway area compared to controls (n = 37). Participants with schizophrenia showed increased sway area following the removal of visual input, while this pattern was absent in controls. Examination of DFA revealed decreased complexity of postural sway and abnormal changes in complexity upon removal of visual input in individuals with schizophrenia. Additionally, less complex postural sway was associated with increased symptom severity in participants with schizophrenia. Given the critical involvement of the cerebellum and related circuits in postural stability and sensorimotor integration, these results are consistent with growing evidence of motor, cerebellar, and sensory integration dysfunction in the disorder, and with theoretical models that implicate cerebellar deficits and more general disconnection of function in schizophrenia.     

Mechanobiology of engineered cartilage cultured under a quantified fluid-dynamic environment

Natural cartilage remodels both in vivo and in vitro in response to mechanical forces and hence mechanical stimulation is believed to have a potential as a tool to modulate extra-cellular matrix synthesis in tissue-engineered cartilage. Fluid-induced shear is known to enhance chondrogenesis on animal cells. A well-defined hydrodynamic environment is required to study the biochemical response to shear of three-dimensional engineered cell systems. We have developed a perfused-column bioreactor in which the culture medium flows through chondrocyte-seeded porous scaffolds, together with a computational fluid-dynamic model of the flow through the constructs' microstructure. A preliminary experiment of human chondrocyte growth under static versus dynamic conditions is described. The median shear stress imposed on the cells in the bioreactor culture, as predicted by the CFD model, is 3 × 10⁻³ Pa (0.03 dyn/cm²) at a flow rate of 0.5 ml/min corresponding to an inlet fluid velocity of 44.2 μm/s. Providing a fluid-dynamic environment to the cells yielded significant differences in cell morphology and in construct structure. Received: 22 December 2001 / Accepted: 18 February 2002     

Effect of chlorpromazine and trifluoperazine on cytoskeletal components and mitochondria in cultured mammalian cells

Antipsychotic drugs such as chlorpromazine and trifluoperazine have been implicated to mediate their action by inhibiting calmodulin, the general calcium regulatory protein in eukaryotic cells. We observed that both these drugs were cytotoxic to different mammalian cell types at concentrations two- to three-fold lower than those required to inhibit calmodulin-dependent phosphodiesterase activity. These drugs also caused shrinkage and rounding of chicken embryo fibroblast cells without affecting any of the cytoskeletal components, viz. microtubules, microfilaments and intermediate filaments. However, at physiological concentrations of these drugs, a major change was observed in mitochondria which assumed rounded and swollen shapes and concentrated towards the perinuclear region of cells. These studies provide evidence that in contrast to earlier reports, cytoskeletal components are not the primary targets of these drugs. It is suggested that mitochondria may be one of the first structures to be affected by these drugs and the consequent energy depletion may lead to the other observed effects.