Extrapolating radiation-induced cancer risks from low doses to very low doses.

There is strong evidence that ionizing radiation increases cancer risks at high doses (e.g., >or=1 Gy), and persuasive, if controversial, epidemiological evidence that cancer risks are increased at low doses ( approximately 10 mGy). Discussed here are the issues related to extrapolating radiation risks from low radiation doses to very low doses (

Detection of intracellular granularity induction in prostate cancer cell lines by small molecules using the HyperCyt high-throughput flow cytometry system.

Prostate cancer is a leading cause of death among men due to the limited number of treatment strategies available for advanced disease. Discovery of effective chemotherapeutics involves the identification of agents that inhibit cancer cell growth. Increases in intracellular granularity have been observed during physiological processes that include senescence, apoptosis, and autophagy, making this phenotypic change a useful marker for identifying small molecules that induce cellular growth arrest or death. In this regard, epithelial-derived cancer cell lines appear uniquely susceptible to increased intracellular granularity following exposure to chemotherapeutics. We have established a novel flow cytometry approach that detects increases in side light scatter in response to morphological changes associated with intracellular granularity in the androgen-sensitive LNCaP and androgen-independent PC3 human prostate cancer cell lines. A cell-based assay was developed to screen for small molecule inducers of intracellular granularity using the HyperCyt high-throughput flow cytometry platform. Validation was performed using the Prestwick Chemical Library, where known modulators of LNCaP intracellular granularity, such as testosterone, were identified. Nonandrogenic inducers of granularity were also detected. A further screen of approximately 25,000 small molecules led to the identification of a class of aryl-oxazoles that increased intracellular granularity in both cell lines, often leading to cell death. The most potent agents exhibited submicromolar efficacy in LNCaP and PC3 cells.

Autophagy in intracellular bacterial infection.

Numerous pathogens have developed the capacity to invade host cells to be protected from components of the systemic immune system. However, once in the host cells they utilize sophisticated strategies to avoid the powerful machinery built by the cells to kill invading pathogens. In the last few years cumulative evidence indicates that autophagy is one of the most remarkable tools of the intracellular host cell defense machinery that bacteria must confront upon cell invasion. However, several pathogens subvert the autophagic pathway and, manipulate this process at the molecular level, as a strategy to establish a persistent infection. In this review we have summarized the interaction between autophagy and different bacterial pathogens including those that take advantage of the host cell autophagy, allowing successful colonization, as well as those microorganisms which are controlled by autophagy as part of the innate surveillance mechanism.

Defective intracellular Ca2+ homeostasis contributes to myocyte dysfunction during ventricular remodelling induced by chronic volume overload in rats.

1. Previous studies have demonstrated progressive ventricular hypertrophy, dilatation and contractile depression in response to chronic volume overload. Whether this decompensation was related to intrinsic myocyte dysfunction was not clear. The present study evaluated ventricular myocyte function at critical times during the progression of ventricular remodelling induced by volume overload. 2. Chronic volume overload was induced with an infrarenal aortocaval fistula in rats. Myocyte contraction and intracellular Ca(2+) concentrations ([Ca(2+)](i)) were evaluated using a fura-2 fluorescence and edge detection system. Protein levels of sarcoplasmic reticulum (SR) Ca(2+) transporters were determined by western blots. Progressive ventricular dilatation developed following creation of the fistula. Although myocyte function in 5 week fistula rats was comparable to that of the control group, myocytes from rats 10 weeks post-fistula demonstrated significant depression of cell shortening and peak [Ca(2+)](i). Application of isoproterenol (0.1 micromol/L) was not able to compensate for the functional deficiency in myocytes from 10 week fistula rats. Caffeine (10 mmol/L) induced SR Ca(2+) release, as well as protein expression of SR Ca(2+)-ATPase, and ryanodine receptors were reduced in myocytes obtained from the same group of 10 week fistula rats. 3. These data indicate that the transition to heart failure secondary to chronic volume overload is related to depressed myocyte contractility secondary to altered intracellular Ca(2+) homeostasis.

Intracellular versican expression in mesenchymal spindle cell tumors contrasts with extracellular expression in epithelial and other tumors--a tissue microarray-based study.

Versican is a large chondroitin sulfate proteoglycan that is an integral component of the extracellular matrix protein. It regulates cell proliferation, adhesion, and migration, and is expressed in a variety of normal tissues and tumors. We studied the pattern of versican expression in various epithelial, mesenchymal, neural, and hematopoietic tumors using immunohistochemistry on tissue microarrays. The primary antibody used was mouse monoclonal antibody to versican (clone 8S270, 1:4000, US Biological). Sections from 3 healing wounds were also included to demonstrate versican expression in reactive tissues. The extracellular matrix in all tissues including all tumors (epithelial and nonepithelial) was positive for versican. However, intracellular cytoplasmic expression of versican was seen only in spindle cells, for example, fibroblasts in healing wounds, 11 of 16 (69%) gastrointestinal stromal tumors and 12 of 42 (28%) smooth muscle tumors. Intracellular versican was not seen in any other tumor [0/344 carcinomas (64 breast, 63 prostate, 61 colorectal, 59 lung, 68 ovarian, and 29 thyroid), 0/22 glioblastoma multiforme, 0/46 lymphomas, and 0/21 melanomas]. As versican plays a role in cell proliferation, differentiation, adhesion, and migration, its differential expression in spindle cell tumors may be associated with the differentiation, progression, and spread of these tumors, which is different from epithelial tumors.

Organically modified silica nanoparticles co-encapsulating photosensitizing drug and aggregation-enhanced two-photon absorbing fluorescent dye aggregates for two-photon photodynamic therapy.

We report energy-transferring organically modified silica nanoparticles for two-photon photodynamic therapy. These nanoparticles co-encapsulate two-photon fluorescent dye nanoaggregates as an energy up-converting donor and a photosensitizing PDT drug as an acceptor. They combine two features: (i) aggregation-enhanced two-photon absorption and emission properties of a novel two-photon dye and (ii) nanoscopic fluorescence resonance energy transfer between this nanoaggregate and a photosensitizer, 2-devinyl-2-(1-hexyloxyethyl)pyropheophorbide. Stable aqueous dispersions of the co-encapsulating nanoparticles (diameter < or = 30 nm) have been prepared in the nonpolar interior of micelles by coprecipitating an organically modified silica sol with the photosensitizer and an excess amount of the two-photon dye which forms fluorescent aggregates by phase separation from the particle matrix. Using a multidisciplinary nanophotonic approach, we show: (i) indirect excitation of the photosensitizer through efficient two-photon excited intraparticle energy transfer from the dye aggregates in the intracellular environment of tumor cells and (ii) generation of singlet oxygen and in vitro cytotoxic effect in tumor cells by photosensitization under two-photon irradiation.

Manipulation of dendritic cells for host defence against intracellular infections.

Dendritic cells (DCs) are an important innate immune cell type which is the bridge between innate and adaptive immunity. Mounting experimental evidence suggests that manipulating DCs represents a powerful means to enhance host defence against intracellular infectious diseases. We have developed several strategies to manipulate DCs either in vivo or in vitro for the purpose of enhancing the effect of vaccination or immunotherapeutics. In vivo delivery of transgene encoding GM-CSF (granulocyte/macrophage colony-stimulating factor), a DC-activating cytokine, increases the number and activation status of DCs at various tissue sites and enhances antimicrobial immune responses in murine models. Co-expression or co-delivery of GM-CSF gene transfer vector with an antimicrobial vaccine enhances microbial antigen-specific T-cell responses and immune protection. Murine bone marrow-derived DCs are being manipulated in vitro and exploited as a vaccine delivery system. Transduction of DCs with a virus-vectored tuberculosis vaccine is a powerful way to activate T-cells in vivo. Such genetically modified DC vaccines can be administered either parenterally or mucosally via the respiratory tract.

Postmortem stability of pituitary hormones in the human adenohypophysis.

The hypophysis is embedded in the fossa at the base of skull, having important functions in the hormonal system. The present study investigated its postmortem morphological changes and the stability of adenohypopyseal hormones. The pituitaries were collected at autopsy 6 h to 20 days postmortem and were studied by histology, immunohistochemistry and electron microscopy. To avoid the influence of prolonged brain hypoxia or swelling, subjects who survived not longer than 12 h were examined. Histological changes were seen in the nucleus 6 h after death, followed by cytoplasmic changes, and the cell shapes were hardly identifiable 7 days postmortem. Electron microscopy revealed evident ultra-structural changes 6 h postmortem, involving rough endoplasmic reticulum, Golgi complexes, mitochondria, nuclei and cell membranes. However, secretory granules remained well preserved 7 days postmortem. Immunostaining showed positivities for growth hormone, prolactin, adenocorticotropic hormone, luteinizing hormone and thyroid-stimulating hormone up to 15 days after death. These findings suggest the usefulness of immunohistochemical investigation of the adenohypophysis for estimating the time of death and endocrinologic evaluation in decomposed cadavers.

Anti-cancer drugs: molecular mechanisms of action.

Genetic alterations are responsible for all cancers. These mutations produce, in turn, alterations in key proteins of certain signaling pathways. Amongst the best known and studied alterations related to malignant transformations are those which occur in Ras protein and p53. In most cases mutations in Ras and p53 lead to the appearance of practically most malignant transformations. Mutated Ras genes exist in approximately 20 to 30% of all human cancers. Ras proteins are switches that regulate diverse functions such as cell proliferation, differentiation and apoptosis. Normal p53 expression, also known as the "genome guardian", is a key molecule for suppressing cell proliferation. The great importance of these proteins rests on their intimacy with the events leading to cell proliferation or death. The comprehension of the extent of transformation on Ras and p53, and of the diverse biochemical pathways of intracellular signaling, activated by them, is of extreme importance for the understanding of malignant transformation, as well as its control, through the creation, for example, of new drugs which contribute to the elimination of these cells. To clarify the consequences originated by transformed Ras, p53 and their biochemical interlinks in the different intracellular pathways, besides the possible intervening points and pharmacological controls presently used in combating cancer, are the aims of this review.

Clinical characteristics of subretinal deposits in central serous chorioretinopathy.

PURPOSE: To describe abnormal subretinal material in central serous chorioretinopathy (CSC). DESIGN: Retrospective observational case series. PARTICIPANTS: 168 consecutive patients (336 eyes) with a definite diagnosis of serous foveal detachment attributable to CSC in one or both eyes, on one or more occasions. METHODS: Review of all cases seen during a six-year period. Grading of the amount of subretinal material at presentation as absent, questionable, mild, moderate or severe. RESULTS: Of 168 patients with CSC, 133 (79%) were men and 35 (21%) women. The median age was 45.2 years (range 22-70 yrs). The median duration of symptoms was 2 months (range 0.1-144 months). Subretinal material was found in the symptomatic eye or, in bilateral cases, in the eye that had most recently become symptomatic in 138 patients and in increasing amounts with increasing duration of symptoms (p < 0.001) but unrelated to age or sex. A substantial fraction of the material was shown by optical coherence tomography to be attached to the photoreceptor outer segments of the detached retina. CONCLUSIONS: Increasing amounts of subretinal material are found with increasing duration of symptoms in eyes with CSC. This suggests that early granular deposits may be composed of fragments of photoreceptor outer segments that accumulate when the phagocytosis photoreceptor outer segment material is disrupted by the serous detachment of the retina. Other possible origins that cannot be excluded include plasma proteins excluding from the choriocapillaris, inflammatory debris, and lipid exudate originating from occult choroidal neovascularization secondary to CSC.

Intracellular Abeta42 activates p53 promoter: a pathway to neurodegeneration in Alzheimer's disease.

The amyloid beta-protein (Abeta) ending at 42 plays a pivotal role in Alzheimer's disease (AD). We have reported previously that intracellular Abeta42 is associated with neuronal apoptosis in vitro and in vivo. Here, we show that intracellular Abeta42 directly activated the p53 promoter, resulting in p53-dependent apoptosis, and that intracellular Abeta40 had a similar but lesser effect. Moreover, oxidative DNA damage induced nuclear localization of Abeta42 with p53 mRNA elevation in guinea-pig primary neurons. Also, p53 expression was elevated in brain of sporadic AD and transgenic mice carrying mutant familial AD genes. Remarkably, accumulation of both Abeta42 and p53 was found in some degenerating-shape neurons in both transgenic mice and human AD cases. Thus, the intracellular Abeta42/p53 pathway may be directly relevant to neuronal loss in AD. Although neurotoxicity of extracellular Abeta is well known and synaptic/mitochondrial dysfunction by intracellular Abeta42 has recently been suggested, intracellular Abeta42 may cause p53-dependent neuronal apoptosis through activation of the p53 promoter; thus demonstrating an alternative pathogenesis in AD.

Demonstration of a role for alpha-synuclein as a functional microtubule-associated protein.

Alpha-synuclein is a major constituent of pathological intracellular inclusion bodies, a common feature of several neurodegenerative diseases. Two missense mutations in the alpha-synuclein gene have been identified in confirmed autosomal-dominant familial Parkinson's disease, which segregate with the illness. However, the physiological function of alpha-synuclein remains unknown. After biochemical investigations we have revealed tubulin to be an alpha-synuclein associated/binding protein. Here, we show that alpha-synuclein induces polymerization of purified tubulin into microtubules. Mutant forms of alpha-synuclein lose this potential. The binding site of alpha-synuclein to tubulin is identified, and co-localization of alpha-synuclein with microtubules is shown in cultured cells. To our knowledge, this is the first demonstration of microtubule-polymerizing activity of alpha-synuclein. Now we can see a striking resemblance between alpha-synuclein and tau: both have the same physiological function and pathological features, making abnormal structures in diseased brains known as synucleinopathies and tauopathies. The discovery of a physiological role for alpha-synuclein may provide a new dimension in researches into the mechanisms of alpha-synuclein-associated neurodegenerative diseases.

Intracellular collagen and fibronexus in fibromatosis and other fibroblastic tumors.

Myofibroblasts are mesenchymal cells with combined function and structure for contraction and collagen synthesis. They are found in reparative responses, nodular fasciitis, fibromatosis, and myofibroblastic sarcoma. Ultrastructurally, myofibroblasts are characterized by a specialized cell surface structure called the fibronexus (FNX). In addition, intracellular collagen fibers (ICF) have been described in nodular fasciitis and fibromatosis, but their origin and nature are still controversial. The aim of the present work was, first, to assess the frequency of FNX and ICF in proliferative myofibroblastic conditions compared to diverse mesenchymal tumors with spindle-shaped cells, and, second, to determine what kind of organelles contain ICF and if they are related to phagocytosis or cell synthesis. Forty-two cases of aggressive fibromatosis and 11 of nodular fasciitis (group A) were compared to 82 spindle-cell mesenchymal tumors of diverse nature (group B) by electron microscopy study. The presence and frequency of FNX and ICF was compared in both groups, and the organelles containing ICF were recorded. FNX and ICF were constantly found in group A (69.8 and 84.9%, respectively), and rarely in group B (0 and 5.12%, respectively). Most frequently ICF were contained in tunnels and phagolysosomes, but also were found in Golgi vesicles and cisternae of rough endoplasmic reticulum. In the majority of cases (75%), ICF were similar to collagen fibers of the extracellular space, but in some cases (22.5%), they were in dissimilar stages of fibrogenesis. Fibromatosis and nodular fasciitis are characterized by proliferation of myofibroblasts and constantly show FNX and ICF. These structures are rarely found in other mesenchymal tumors. The ICF are found in organelles of digestion and also in others related to synthesis and transport.

Activated p38MAPK is a novel component of the intracellular inclusions found in human amyotrophic lateral sclerosis and mutant SOD1 transgenic mice.

Cytoskeletal abnormalities with accumulation of ubiquilated inclusions in the anterior horn cells are a pathological hallmark of both familial and sporadic amyotrophic lateral sclerosis (ALS) and of mouse models for ALS. Phosphorylated neurofilaments besides ubiquitin and dorfin have been identified as one of the major components of the abnormal intracellular perikaryal aggregates. As we recently found that p38 mitogen-activated protein kinase (p38MAPK) colocalized with phosphorylated neurofilaments in spinal motor neurons of SOD1 mutant mice, a model of familial ALS, we investigated whether this kinase also contributed to the inclusions found in ALS patients and SOD1 mutant mice. Intense immunoreactivity for activated p38MAPK was observed in degenerating motor neurons and reactive astrocytes in ALS cases. The intracellular immunostaining for activated p38MAPK appeared in some neurons as filamentous skein-like and ball-like inclusions, with an immunohistochemical pattern identical to that of ubiquitin. Intracellular p38MAPK-positive aggregates containing ubiquitin and neurofilaments were also found in the spinal motor neurons of SOD1 mutant mice. Our observations indicate that activation of p38MAPK might contribute significantly to the pathology of motor neurons in ALS.

Morphological and cytochemical characteristics of leukaemic promyelocytes.

Evaluation of cell morphology is usually sufficient to diagnose acute promyelocytic leukaemia (APL). In this chapter we discuss the features of classical hypergranular APL, the APL variant, hyperbasophilic promyelocytic leukaemia, APL with basophil-like granules, acute eosinophilic leukaemia with PML/RARalpha positivity and the morphology of APL cells lacking t(15;17). In addition to morphological examination, cytochemical investigations (peroxidase chloroacetate-esterase, etc.) may help further in defining the cytology of leukaemic cells in APL.