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The molecular mechanisms that drive mammalian cells to the development of cancer are the subject of intense biochemical, genetic and medical studies. But for the present, there is no comprehensive model that might serve as a general framework for the interpretation of experimental data. This paper is an attempt to create a conceptual model of the mechanism of the developing tumorigenic phenotype in mammalian cells, defined as having high genomic instability and proliferative activity. The basic statement in the model is that mutations acquired by tumor cells are not caused directly by external DNA damaging agents, but instead are produced by the cell itself as an output of a Mutator Response similar to the bacterial "SOS response" and characterized by the initiation of error-prone cell cycle progression and an elevated rate of mutation. This response may be induced in arrested mammalian cells by intracellular and extracellular proliferative signals combined with blocked apoptosis. The mutant cells originated by this response are subjected to natural selection via apoptosis and turnover. This selection process favors the survival of cells with high proliferative activity and the suppression of apoptosis resulting in the long run in the appearance of immortalized cells with high proliferative activity. Either a sustained stressful environment accompanied by continuing apoptotic cell death, or replicative senescence, provides conditions suitable for activation of the Mutator Response, namely the emergence of arrested cells with blocked apoptosis and the induction of proliferative signal. It also accelerates the selection process by providing continuing cell turnover. The proposed mechanism is described at the level of involved metabolic pathways and proteins and substantiated by the related experimental data available in the literature.

In infections, microbial components provide signals that alert the immune system to danger and promote the generation of immunity. In the absence of such signals, there is often no immune response or tolerance may develop. This has led to the concept that the immune system responds only to antigens perceived to be associated with a dangerous situation such as infection. Danger signals are thought to act by stimulating dendritic cells to mature so that they can present foreign antigens and stimulate T lymphocytes. Dying mammalian cells have also been found to release danger signals of unknown identity. Here we show that uric acid is a principal endogenous danger signal released from injured cells. Uric acid stimulates dendritic cell maturation and, when co-injected with antigen in vivo, significantly enhances the generation of responses from CD8+ T cells. Eliminating uric acid in vivo inhibits the immune response to antigens associated with injured cells, but not to antigens presented by activated dendritic cells. Our findings provide a molecular link between cell injury and immunity and have important implications for vaccines, autoimmunity and inflammation.

The advent of gene chips has led to a promising technology for cell, tumor, and cancer classification. We exploit and expand the methodology of recursive partitioning trees for tumor and cell classification from microarray gene expression data. To improve classification and prediction accuracy, we introduce a deterministic procedure to form forests of classification trees and compare their performance with extant alternatives. When two published and commonly used data sets are used, we find that the deterministic forests perform similarly to the random forests in terms of the error rate obtained from the leave-one-out procedure, and all of the forests are far better than the single trees. In addition, we provide graphical presentations to facilitate interpretation of complex forests and compare our findings with the current biological literature. In addition to numerical improvement, the main advantage of deterministic forests is reproducibility and scientific interpretability of all steps in tree construction.

Ricin is a heterodimeric protein produced in the seeds of the castor oil plant (Ricinus communis). It is exquisitely potent to mammalian cells, being able to fatally disrupt protein synthesis by attacking the Achilles heel of the ribosome. For this enzyme to reach its substrate, it must not only negotiate the endomembrane system but it must also cross an internal membrane and avoid complete degradation without compromising its activity in any way. Cell entry by ricin involves a series of steps: (i) binding, via the ricin B chain (RTB), to a range of cell surface glycolipids or glycoproteins having beta-1,4-linked galactose residues; (ii) uptake into the cell by endocytosis; (iii) entry of the toxin into early endosomes; (iv) transfer, by vesicular transport, of ricin from early endosomes to the trans-Golgi network; (v) retrograde vesicular transport through the Golgi complex to reach the endoplasmic reticulum; (vi) reduction of the disulphide bond connecting the ricin A chain (RTA) and the RTB; (vii) partial unfolding of the RTA to render it translocationally-competent to cross the endoplasmic reticulum (ER) membrane via the Sec61p translocon in a manner similar to that followed by misfolded ER proteins that, once recognised, are targeted to the ER-associated protein degradation (ERAD) machinery; (viii) avoiding, at least in part, ubiquitination that would lead to rapid degradation by cytosolic proteasomes immediately after membrane translocation when it is still partially unfolded; (ix) refolding into its protease-resistant, biologically active conformation; and (x) interaction with the ribosome to catalyse the depurination reaction. It is clear that ricin can take advantage of many target cell molecules, pathways and processes. It has been reported that a single molecule of ricin reaching the cytosol can kill that cell as a consequence of protein synthesis inhibition. The ready availability of ricin, coupled to its extreme potency when administered intravenously or if inhaled, has identified this protein toxin as a potential biological warfare agent. Therapeutically, its cytotoxicity has encouraged the use of ricin in 'magic bullets' to specifically target and destroy cancer cells, and the unusual intracellular trafficking properties of ricin potentially permit its development as a vaccine vector. Combining our understanding of the ricin structure with ways to cripple its unwanted properties (its enzymatic activity and promotion of vascular leak whilst retaining protein stability and important immunodominant epitopes), will also be crucial in the development of a long awaited protective vaccine against this toxin.

Unlabelled: During interplanetary flights the nuclei of all of a crew member's cells could be traversed by at least one high-LET (Linear Energy Transfer) cosmic-ray particle. In mammalian cells irradiated in vitro about 1 in 10,000 of the surviving cells traversed by heavy particles is transformed to malignancy or mutated. What, if anything, happens to the remaining >99% of surviving cells? A retrospective analysis of archived data and samples from heavy-ion irradiation experiments with cultured human cells in vitro indicated that heavy ions caused a dose- and LET-dependent reduction in growth rates of progeny of irradiated cells, based on colony-size distributions. The maximum action cross section for this effect is between 100 and 300 microm2, at least as large as the cell nuclear area and up to 3 times the cross section for cell killing. Thus, heritable slow growth is the most prevalent effect of high-LET radiations on cultured animal cells, which may have implications for crew health during deep space travel., Disclaimer: The views expressed in this article are those of the author(s) and do not necessarily reflect the views or policies of the USEPA.

The ionizing radiation induced bystander effect is initiated by damage to a cellular molecule which then gives rise to a signal exported to other cells. The nature of this damage is considered with the understanding that it may not be the same as that responsible for the traditional cellular effects of radiation. Consideration is give to amounts of endogenous damage and to radiation yields of the various candidate lesions.

Complex multicellular organisms have evolved mechanisms to ensure that individual cells follow their proper developmental and somatic programs. Tumorigenesis, or uncontrolled cellular proliferation, is caused by somatic mutations to those genetic constraints that normally operate within a tissue. Genes involved in DNA repair and apoptosis are particularly instrumental in safeguarding cells against tumorigenesis. In this paper, we introduce a stochastic framework to analyse the somatic evolution of cancer initiation. Within this model, we study how apoptosis and DNA repair can maintain the transient stability of somatic cells and delay the onset of cancer. Focusing on individual cell lineages, we calculate the waiting time before tumorigenesis in the presence of varying degrees of apoptosis and DNA repair. We find that the loss of DNA repair or the loss of apoptosis both hasten tumorigenesis, but in characteristically different ways., (Copyright 2002 Elsevier Science Ltd.)

Exacerbation of hypoxic injury after restoration of oxygenation (reoxygenation) is an important mechanism of cellular injury in transplantation and in myocardial, hepatic, intestinal, cerebral, renal, and other ischemic syndromes. Cellular hypoxia and reoxygenation are two essential elements of ischemia-reperfusion injury. Activated neutrophils contribute to vascular reperfusion injury, yet posthypoxic cellular injury occurs in the absence of inflammatory cells through mechanisms involving reactive oxygen (ROS) or nitrogen species (RNS). Xanthine oxidase (XO) produces ROS in some reoxygenated cells, but other intracellular sources of ROS are abundant, and XO is not required for reoxygenation injury. Hypoxic or reoxygenated mitochondria may produce excess superoxide (O) and release H(2)O(2), a diffusible long-lived oxidant that can activate signaling pathways or react vicinally with proteins and lipid membranes. This review focuses on the specific roles of ROS and RNS in the cellular response to hypoxia and subsequent cytolytic injury during reoxygenation.

Information has been provided in this chapter on the fundamentals of thermal sterilization processing. The derivation of various equations presented in the text was not discussed; the reader is referred to the references cited for this information. Emphasis was placed on the need for good manufacturing practice and for control of presterilization bioburden. These considerations will have the greatest impact on product quality in the long run. An attempt was also made to dispel the notion that sterilization processes always require the most rigorous biological challenge for validation. Finally, parametric or process control release of terminally sterilized products was discussed. Parametric release is a concept that will find greater application in areas other than most heat sterilization as the industry continues to derive full benefit from validated processes.

The cytolethal distending toxins (CDTs) are a newly discovered family of bacterial protein toxins with the unique ability to interfere with the cell cycle, causing irreversible cell cycle arrest and consequently death of the target cells. CDTs are encoded by three linked genes (cdtA, cdtB and cdtC) and are produced by a variety of Gram negative bacteria. The mechanism of action of this toxin family only now begins to be elucidated. CDTs are internalized by endocytosis and require an intact Golgi complex to exert their cytotoxic activity. The CdtB component was shown to have functional homology with the mammalian deoxyribonuclease I (DNase I) and the induction of cell cycle arrest in mammalian cells mimicked that induced by DNA damaging agents, suggesting that DNA is the cellular target. Still there are many issues that need to be clarified, such as identification of the function(s) of CdtA and CdtC, characterization of the receptor(s), understanding of the final steps of the internalization pathway and localization of the active component. This review focuses mainly on the effect of CDTs on mammalian cells, highlighting the questions that remain to be answered regarding their molecular mode of action.

The genetic response of human cells to sublethal thermal injury was assessed by gene expression profiling, using macroarrays containing 588 complementary known genes. At 1, 4, 8, and 24 h following thermal injury, RNA was isolated, and a cDNA copy was generated incorporating (33)P and hybridized to Atlas arrays. About one-fifth of the genes on the membrane exhibited a significant elevation or depression in expression (>/=2-fold) by 4 h posttreatment. Genes for heat shock proteins (HSPs) were upregulated as well as genes for transcription factors, growth regulation, and DNA repair. Cluster analysis was performed to assess temporal relationships between expression of genes. Translation of mRNA for some expressed genes, including HSP70 and HSP40, was corroborated by Western blotting. Gene expression profiling can be used to determine information about gene responses to thermal injury by retinal pigment epithelium cells following sublethal injury. The induction of gene expression following thermal injury involves a number of genes not previously identified as related to the stress response.

Besides competing with algae for light and mineral nutrients (i.e. N, P, etc.), the root system of Acorus tatarinowii excretes some chemical substances, which injure and eliminate alga cells, to inhibit the growth of the algae. When the algae cells were treated in "A. tatarinowii water", some of the chlorophyll a were destroyed and the photosynthetic rate of algae decreased markedly and the ability of alga cells to deoxidize triphenyltetrazolium chloride (TTC) reduced greatly. Then alga cells turned from bright red to bluish green under fluorescence microscope. These showed that the allelopathic effects of A. tatarinowii on algae were obvious and planting A. tatarinowii can control some green algae. The experiment on the extractions of the secretions of the root system showed that the inhibitory effect had a concentration effect. If the concentration of the root secretion was below 30 microliters/disc, the inhibitory rate was negative; if it was over 45 microliters/disc, the inhibitory rate was positive. This proved that the influence of the root secretion on the same acceptor was a kind of concentration effect. When the concentration of the root secretion was low, it promoted the growth of algae; when the concentration reached a definite threshold value, it restrained the growth of algae. In present case, the threshold value was between 30 microliters/disc and 45 microliters/disc.

Multiple organ failure remains the major cause of death in critically ill patients. In view of therapeutic strategies, current research activities focus on the cellular response to different kinds of cellular stress (hypoxia, oxidative damage and mechanical distress) in the pathogenic sequelae of organ failure. The cellular stress reactions are characterized by induction of adaptive programs of gene expression (e.g. acute phase proteins, heat shock proteins, hypoxia-associated proteins) to protect the cells from energy depletion and cell death. Generally, the mitochondria are early indicators of cellular stress showing a loss of cytochrome c and a breakdown of the transmembrane potential. Shortage of ATP and decrease of pH can be observed in the cytoplasm which leads to a disintegration of the cytoskeleton. As a consequence, the cell becomes spherical and separates from the surrounding cells. Depending on the acuity of the stressor, the cell dies due to necrosis or apoptosis. Dysregulation of the balance of apoptosis and necrosis in different organs seems to be an important mechanism in the development of organ failure. New insights into the cellular mechanisms during organ dysfunction promote the development of new diagnostic (e.g., optical and spectroscopic) and pharmacological tools leading to a better prevention and therapy of organ failure.

The paper describes a mathematical model for determining the likelihood of radiation-induced complications in the tissue as a function of the number of surviving cells. It is suggested the irradiated tissue cannot be regarded as a structureless point set of cells and that there are repairable and nonrepairable spatial configurations formed by surviving tissue cells. The probability of none tissue radiation complications may be considered is that of formation of a repairable structure from the surviving tissue cells. The paper shows why the radiation effect of radiation may be coupled (graded) in units of integral cellularity. Recurrent equations have been derived for calculating the number of repairable structures in relation to the conditions of tissue radiation. The values of a number of repairable structures as a function of an area and single dose of radiation are calculated as an example.

This unit describes two methods of in situ hybridization: one uses an ³⁵S-labeled oligonucleotide probe and the other uses a digoxigenin-labeled oligonucleotide probe on frozen, cryostat-sectioned samples. These methods allow detection of the physical distribution and expression levels of target mRNA. Protocols are also included for labeling the probes and preparing the sample material.

Toxicant-induced damage to cells (cytotoxicity) can depress cell growth, compromise intracellular metabolic processes, and/or cause loss of cell viability. Methods that indicate these cytotoxic changes following toxicant exposures are provided, including [³H]thymidine uptake to measure cell growth, MTT dye conversion to detect changes in metabolic activity, and trypan blue uptake to indicate loss of cell viability.

The heat shock protein family of proteins include a number of proteins of similar sizes that are inducible by a wide variety of "stress" stimuli including heat shock, ischemia, toxic metals, amino acid analogs, and some chemotherapeutic and anesthetic chemicals. The functions of these proteins are diverse but are generally associated with molecular chaperoning of proteins. This unit contains a wide variety of protocols for analyzing expression of this family at the transcriptional and translational level: northern blot hybridization, in situ hybridization, metabolic labeling and SDS-PAGE, and immunoblotting.

Confocal microscopy allows visualization of optical sections of material labeled with fluorescence or reflecting probes. By excluding light from planes above and below the plane of focus it is possible to obtain sharp images of objects deep within sections. Sections can be combined to construct three-dimensional images. This unit provides an overview and introduction to confocal microscopy.

Fanconi anemia is a rare autosomal recessive disease characterized by multiple congenital abnormalities, bone marrow failure, and cancer susceptibility. The mean age of onset of anemia is 8 years, and the mean survival is 16 years. Death usually results from complications of bone marrow failure. Considerable progress in Fanconi anemia research has resulted from the recent identification and cloning of three Fanconi anemia genes. The current review describes the structure and function of the Fanconi anemia genes and describes the role of the encoded Fanconi anemia proteins in a cellular pathway controlling chromosome stability.

The mechanisms of cellular damage caused by infectious and inflammatory processes are complex and are still not fully understood. There is, however, a consensus that reactive oxygen species (ROS) generated by phagocytes migrating to injured tissues might be the main agents responsible for cellular damage in inflammatory processes. However, because both activated phagocytes and catalase-negative, peroxide-producing, toxigenic bacteria (Streptococci, Clostridiae) secrete a near-identical array of proinflammatory agonists, including reactive oxygen species (ROS), and because these microbial species might kill their targets by a synergism among several of their secreted enzymes (a multicomponent system), we postulated that activated phagocytes might also function in the same way. Using radiolabeled targets, in culture, we demonstrated that subtoxic amounts of a variety of oxidants (H2O2, radicals produced by xanthine-xanthine-oxidase, peroxyl radical, NO) acted synergistically with subtoxic amounts of a large series of membrane-perforating agents (microbial hemolysins, phospholipases, fatty acids, cationic proteins, proteinases, bile salts, the attack complex of complement, the xenobiotics, lindane, ethanol, methanol) to kill cells in culture and to release large amounts of arachidonic acid and metabolites. Membrane perforators might act primarily to overcome the potent antioxidant systems present in all mammalian cells and scavengers of ROS and inhibitors of the additional agonists might act to abolish the synergism among ROS and the membrane-damaging agents. It is also proposed that protection against tissue damage in vivo should also include 'cocktails' of appropriate antagonists. It is enigmatic that those publications which do describe both in-vitro and in-vivo models proposing that a synergism among a multiplicity of agonists might truly represent the mechanisms by which tissues are injured, in vivo, are hardly ever quoted in the current literature.

Three stages of development in cellular pathology are analyzed. Evaluation of important achievements in the past which permitted the advance to the present stage in cellular pathology is given. New methods in experimental pathology resulted in the appearance of a new direction--molecular morphology and molecular cell pathology. The basic features of this direction and new problems to be resolved are formulated.

According to a considerable amount of publications apoptosis plays an important role for radio- and chemotherapy. The most important results related to this issue will be described in 2 independent articles, covering the following topics: Part I: I. definition, morphology, biochemical processes, II. clinical relevant detection assays, III. signal transduction. Part II: significance of apoptosis for radio- and chemotherapy.

The cell death is a natural physiological process that occurs in every living organism. It is clear that programmed cell death--apoptosis--is an important mechanism maintaining cell number in a multicellular organism. This review summarises recent progress in the field of apoptosis. Extracellular signals, such as various growth factors and antigen Fas ligand can trigger apoptosis via cell surface receptors. Within the cell the tumor suppressor gene p53 and oncogenes c-myc, c-fos and c-jun tend to activate apoptosis, while other genes such as most members of the bcl-2 family, tend to suppress it. Many of these signals regulate a family of cysteine proteases--related to interleukin 1 beta converting enzyme (ICE)--caspases--which play a crucial role in apoptosis. Many factors that affect apoptosis also affect the cell cycle. For example, p53 appears to be an important mediator of both apoptosis and cycle arrest. If DNA damage is repaired during cycle arrest, the cell survives. Special attention is paid to the abnormalities in the regulation of apoptosis that may contribute to different pathogenic processes.

The glycolytic protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH) appeared to be an archtypical protein of limited excitement. However, independent studies from a number of different laboratories reported a variety of diverse biological properties of the GAPDH protein. As a membrane protein, GAPDH functions in endocytosis; in the cytoplasm, it is involved in the translational control of gene expression; in the nucleus, it functions in nuclear tRNA export, in DNA replication, and in DNA repair. The intracellular localization of GAPDH may be dependent on the proliferative state of the cell. Recent studies identified a role for GAPDH in neuronal apoptosis. GAPDH gene expression was specifically increased during programmed neuronal cell death. Transfection of neuronal cells with antisense GAPDH sequences inhibited apoptosis. Lastly, GAPDH may be directly involved in the cellular phenotype of human neurodegenerative disorders, especially those characterized at the molecular level by the expansion of CAG repeats. In this review, the current status of ongoing GAPDH studies are described (with the exception of its unique oxidative modification by nitric oxide). Consideration of future directions are suggested.

Within a homogeneous population of cells, the tendency of individual cells to become diseased is not 'homogeneous'. As a general concept of disease, the 'liability theory' describes such a tendency (cell liability) as a quantitative characteristic following a continuous distribution. This suggests that there are always some cells that are more liable than the rest and that the liability values of cells or liability-associated cell characteristics can be assessed quantitatively. By defining the more or extremely liable cells through a critical value or 'threshold', the liability theory hypothesizes that these cells may play a significant role in determining or affecting the overall disease liability of a cell population; the more such cells are present, the more liable the cell population is, as a whole, to disease. Furthermore, it proposes that the origin and development of disease may be fundamentally relevant to this kind of intrapopulational variation in cell liability. The liability theory attempts to provide a theoretical framework for studying the 'cell population' mechanism of disease, or cell population pathology. Also, it may serve as a working model for analysis and assessment of cell population liabilities for various diseases.

A holistic approach to tumorigenesis is proposed. The main element of the model is the existence of dynamic cellular network. This network comprises a molecular and an energetistic structure of a cell connected through the multidirectional flow of information. The interactions within dynamic cellular network are complex, stochastic, nonlinear, and also involve quantum effects. From this non-reductionist perspective, neither tumorigenesis can be limited to the genetic aspect, nor the initial event must be of molecular nature, nor mutations and epigenetic factors are mutually exclusive, nor a link between cause and effect can be established. Due to complexity, an unstable stationary state of dynamic cellular network rather than a group of unrelated genes determines the phenotype of normal and transformed cells. This implies relativity of tumor suppressor genes and oncogenes. A bifurcation point is defined as an unstable state of dynamic cellular network leading to the other phenotype-stationary state. In particular, the bifurcation point may be determined by a change of expression of a single gene. Then, the gene is called bifurcation point gene. The unstable stationary state facilitates the chaotic dynamics. This may result in a fractal dimension of both normal and tumor tissues. The co-existence of chaotic dynamics and complexity is the essence of cellular processes and shapes differentiation, morphogenesis, and tumorigenesis. In consequence, tumorigenesis is a complex, unpredictable process driven by the interplay between self-organisation and selection.

Electron probe X-ray microanalysis has been used for the last 25 years by biologists to obtain information about the distribution of elements at the cell and tissue level. During this period, progress has mainly been made through the development of more adequate techniques for specimen preparation (mainly low temperature techniques) and quantitative analysis, so that accurate analysis of the physiologically important cellular ions can be carried out. Use of in vitro systems and cell cultures may further increase the number of problems to which X-ray microanalysis can be applied. Among the numerous applications of X-ray microanalysis in cell biology and cell pathology, applications in the areas of epithelial ion transport, the role of calcium in secretory and contractile cells, and the role of ions in cell proliferation and cancer are discussed in more detail.

Laser-induced stress waves can be generated by one of the following mechanisms: optical breakdown, ablation, or rapid heating of an absorbing medium. These three modes of laser interaction with matter allow the investigation of cellular and tissue responses to stress waves with different characteristics and under different conditions. The effects of stress waves on cells and tissues can be quite disparate. Stress waves can fracture tissue, kill cells, decrease cell viability and increase the permeability of the plasma membrane. They can induce deleterious effects during medical procedures of high power, short pulse lasers or, alternatively, may facilitate new therapeutic modalities, such as drug delivery and gene therapy. This review covers the generation of laser-induced stress waves and their effects on cell cultures and tissue.

The objective of this study was to investigate the in vivo relationship between plasma free fatty acid (FFA)/albumin molar ratio and indicators of cellular damage. A case series study was carried out in 20 geriatric patients in a stable clinical condition. Their plasma albumin concentration was in the range 26-42 g l-1. There was a significant positive correlation between the FFA/albumin ratio and (a) reticulocyte count (r = 0.61, p = 0.006), (b) lactate dehydrogenase activity (r = 0.69, p = 0.002), and (c) haptoglobin concentration (r = 0.46, p = 0.05). The haemoglobin concentration was inversely related to relative reticulocyte count (r = -0.55, p = 0.01). Absolute and relative reticulocyte counts were positively associated (r = 0.92, p < 0.0001). The results are in accordance with the contention that a high FFA/albumin ratio in vivo may elicit cellular damage. Further studies are required to elucidate to what extent a high FFA/albumin ratio might be causally related to diseases.

In diseases leading to massive acute cell damage, e.g., myocardial infarction or spontaneous inflammation, increased amounts of hydroperoxides of unsaturated fatty acids (LOOH) are found. An even higher production of LOOH is observed in homogenized tissue. If cells are injured, dormant lipoxygenases (LOX) are inevitably activated. They oxidize unsaturated membrane fatty acids to LOOH. This process involves not only arachidonic acid - as tacitly assumed up to now - but also linoleic acid. LOOH are decomposed to chemically highly reactive species, some of which were previously unknown (e.g, alpha-hydroxyaldehydes). LOO. radicals can also transform any molecule with a double bond to an epoxide. Therefore, epoxides are found in injured tissue. The same degradation products of hydroperoxides have been observed in elevated amounts in acute cell injury and in chronic diseases, e.g., atherosclerosis, psoriasis, and rheumatoid diseases. Therefore, we conclude that in these cases too, increased generation of hydroperoxides is caused by gradual cell injury liberating lipoxygenases.

Mitochondrial function is closely linked to the maintenance of mitochondrial integrity. During short-term anoxia, ion-transport systems in the inner membrane are inhibited to protect against loss of the promotive force and associated osmotic imbalance that can cause irreversible loss of mitochondrial integrity and function. In two models of chemically induced mitochondrial failure, a prostaglandin B1 derivative, di-calciphor, protected against mitochondrial failure and prevented cell death. Characteristics were similar to those observed in mitochondria during short-term anoxia. Thus, the results indicate that di-calciphor may represent a new type of mitochondrial protectant that inhibits ion transport and thus slows the loss of osmotic stability and delays mitochondrial dysfunction under traumatic and toxicologic conditions.

All kinds of hypotheses have been proposed to explain the mechanism of tumorigenesis. Up until now, we have not had any generally acknowledged model that helps us understand the process. In this paper, I propose a dynamic model of how tumor cells escape the supervision of the immune system; I also examine the possibility that tumorigenesis is a two-stage process. Finally, I suggest that our understanding of the molecular level of tumorigenesis be reappraised.

Background and Purpose: Vasogenic brain edema is a frequent complication of ischemic stroke. The mechanism of the blood-brain barrier opening that underlies the edema formation is poorly understood. In the present study we examined the response of endothelial cells cultured from adult human brain to thrombogenic and fibrinolytic factors that possibly accumulate in the occluded vascular segments in ischemic stroke., Methods: The changes in the morphology of cultured human brain microvascular endothelial cells were observed by phase-contrast light microscopy and quantified with computerized morphometry., Results: Active proteases (eg, thrombin, plasmin, urokinase) as well as heparin and protamine, but not fibrinogen and antithrombin III, produced significant changes in endothelial cell morphology. Two shape patterns of contraction were observed: protamine treatment resulted in rounded cells with a decrease in both cell perimeter and area, whereas all other agents induced spiderlike cell morphology with increased perimeter and reduced area. The rate of contraction was dose dependent, and at comparable enzyme concentrations plasmin produced faster contraction than thrombin. The observed changes were reversed 3 hours after abrogating the treatment., Conclusions: In an in vitro model we have demonstrated that factors involved in thrombus formation and dissolution induce endothelial cell contraction, which could affect focally the permeability of the blood-brain barrier by opening paracellular avenues between endothelial cells in vivo. Thus, the genesis of brain edema in thromboembolic stroke or occasionally during fibrinolytic therapy can be attributed in part to the contact of these factors with the microvascular endothelium.

The effect of intracellular ion deregulation, particularly of [Ca2+], on the events following acute cell injury and the progression of change from initiation (reversible) to maintenance (reversible-irreversible) phases and finally to cell death has been the major thrust of experimentation in our laboratory for over 20 years. Cell death, which plays an important role in both normal and pathological phenomena, has been classified into two principal types, accidental and programmed. Recent exploration of programmed cell death (or apoptosis) has revealed extensive data showing it is an important mechanism for the normal maintenance and also differentiation of a variety of cell types and organs. From the results from our laboratory and those of others, we continue to expand and refine our working hypothesis: deregulation of [Ca2+] results in a number of phenomena from activation of signaling mechanisms and alterations in cellular structure to alterations in gene expression, all of which contribute to or play a critical role in cellular toxicity, including carcinogenesis and cell death. Therefore, although much more experimentation is needed to clarify some of these phenomena, the implications of such data for understanding the mechanisms and processes involved in carcinogenesis and the chemotherapeutic killing of cancer cells are extremely exciting. These relationships between [Ca2+], cell injury, and cell death are briefly reviewed here within the framework of our hypothesis.

A modification of the cell block technique, useful in processing material obtained by fine needle aspiration (FNA), is described. Four hundred six aspirates, obtained from 333 consecutive patients, were studied after immediate fixation in 4% buffered paraformaldehyde. Conventional histochemical and immunohistochemical staining methods were used. Histologic verification of the cytologic diagnoses made by FNA was possible in 67 cases. The overall accuracy was 97%, with a sensitivity of 95% and specificity of 100%. A major disadvantage of the cell block technique is time. Therefore, even if this technique increases the accuracy of cytologic diagnosis, its routine use is impractical because the delay in diagnosis when compared with smears may be considerable. The cell block technique is a valuable method, particularly when immunohistochemical staining for a battery of markers is required.

Release of intracellular enzymes to the extracellular space is a marker of cell damage in various diseases, e.g. liver, heart and muscle diseases. In the normal state the plasma membrane is impermeable to enzymes, and enzyme release, therefore, indicates a severe change of the membrane integrity. This review deals with the present knowledge about cellular changes leading to enzyme release, which may be caused either by energy depletion, e.g. in ischemia or shock, or by a direct membrane damage as caused by various toxins and inflammatory products. Inhibition of the energy metabolism results in ATP depletion leading to fluxes of Na+, K+ and Cl- down their gradients across the membrane and swelling of the cell. Subsequently Ca2+ leak into the cell activating phospholipases and the formation of eicosanoids, affecting the cytoskeleton and, perhaps, activating the formation of oxidants. The exact "point of no return" is not known but an uncontrolled Ca2+ activity in the cell probably has an important role in initiating the irreversible changes. The result of these reactions and probably other unknown reactions as well is damage to the membrane. This is evident morphologically at first by the formation of blebs that appears in the reversible phase, and later on by rupturing of the membrane, a sign of irreversible damage. A very small part of the enzyme release may occur in the reversible phase when blebs detach with resealing of the membrane, but the substantial part of enzyme release occurs as a result of irreversible cell damage when ATP has decreased to a low level and a serious disruption of the membrane integrity has taken place. All the secondary affections of the membrane during energy depletion may also occur as a primary direct membrane damage that more or less may affect the energy metabolism secondarily. The cell damage and enzyme release after some types of direct membrane damage is almost independent of the cellular energy metabolism whereas other types of direct membrane damage are counteracted by the cell by energy consuming reactions and, therefore, the final cell damage is a concerted action of the direct membrane damage and the energy depletion. This also means that a direct membrane damage may be more severe for the cell in energy depleted states than in the normal state. As in energy dependent cell damage the substantial part of enzyme release after a direct membrane damage is due to irreversible cellular changes. It appears that although the knowledge of the molecular basis of cell damage and enzyme release has grown there are still many questions to be answered about these complex processes.

A brief review of the historical events leading to developments of image analysis and flow cytometry is presented. The principles, accomplishments, and pitfalls of both techniques of quantitation of cell components and specific applications to markers potentially useful in cancer chemoprevention are discussed.