The gram positive bacillus, Listeria monocytogenesis a contaminant of the food we eat and although it is a common pathogen in farm animals L. monocytogenesdoes not normally infect humans. However, immunocompromised individuals, infants and the elderly are susceptible to infection. The bacteria, taken into membrane bound phagosomes, use a thiol-activated cytolysin listeriolysin O to disrupt the membrane (LLO) and gain access to the cell cytoplasm. Subsequent actin polymerizations on the bacterial surface induce motility in the cell and long cytoplasmic protrusions at the cell surface that spread bacteria to neighboring cells.

L. monocytogenesenters cells by phagocytosis. By using short infection times of 5 min or less, it was possible to examine early interactions between J774 cells and L. monocytogenes.This study shows that during the early stages of internalization the bacteria are able to modify the protein composition of the forming phagosome membrane.

Efavirenz (Sustiva™) is a non-nucleoside reverse transcriptase inhibitor (NNRTI) for the treatment of HrV-infected patients and has been in the market since 1998. Efavirenz exists as three different polymorphs (Forms I, II, III) and a nonstochiometric heptane solvate (Form IV). The current synthetic process for Efavirenz yields Form IV which is converted to the desired Form I polymorph by heating. One of the concerns for any drug substance is whether a consistent crystal form is produced in order to insure consistent physical and chemical properties, as well as to safeguard pharmacokinetic profiles. The sensitivity and resolution of single-crystal x-ray diffraction, the traditional method employed to determine unit cell parameters of drug compounds in development, are limited for Efavirenz Form IV because of the small crystallite size of the material. Furthermore, Efavirenz Form IV materials, depending on the process conditions, have slightly different powder diffraction patterns in terms of intensity when collected on standard in-house diffractometers.

Microscopy, microdissection, and analytical chemistry techniques were used to localize and identify drug and drug metabolites in specimens from drug-treated rats. During a toxicology study in rats of a drug candidate, concretions (stones) were found in the common bile duct, and pigmented material was found in hepatocytes and liver macrophages after long-term exposure to the drug. Because a recent study showed the effective use of microscopy and mass spectrometry (MS) to identify drug metabolite in tissue sections, we used similar methods to confirm that pigmented material in liver cells was drug metabolite. Energy dispersive x-ray spectroscopy (EDS) suggested that drug and/or metabolite were also present in stones.

Pigmented hepatocytes and macrophages in frozen sections of unfixed liver exhibited fluorescence similar to that of parent drug. When excited by UV illumination, these cells fluoresced the same color blue as the bulk drug (Fig. 1).

Cellular oxidative stress, a common mechanism of drug-induced toxicity, is a result of the formation of reactive oxygen species (ROS) in response to chemical stimuli. An endpoint of ROS production is lipid peroxidation, which can in turn lead to disruption of cellular membranes, loss of mitochondrial function, protein oxidation and DNA damage. This toxicity can be organ-specific due to the varying capacities of tissues to handle oxidative events. Liver is particularly sensitive to the effects of oxidative stress, and hepatic toxicity is seen clinically. HepG2 cells are an immortalized human hepatoma cell line used as an in vitro model for mammalian hepatotoxicity studies. The purpose of this study was to characterize the effects of chemically induced oxidative stress in this cultured cell model.

HepG2 cells were grown to subconfluence in poly-L-lysine coated 4-well LabTek™ II chambered coverglasses (Nalge Nunc International).

The insulin-sensitizing agent troglitazone (Rezulin) has been shown to cause severe hepatotoxicity and liver failure. While the toxic mechanism is still unknown, a component may be oxidative stress. We have demonstrated that increased lipid peroxidation, decreased glutathione levels, and collapse in mitochondrial membrane potential occurred following exposure of cultured hepatocytes to troglitazone. These hallmarks of oxidative stress can manifest morphologically in several ways including damage to cellular membranes, mitochondrial abnormalities, and cell death. The purpose of this study was to assess morphologic changes caused by exposure to troglitazone or cumene hydroperoxide (CH), a compound known to induce oxidative stress.

HepG2 and Novikoff hepatoma cells were grown on poly-L-lysine coated 6-well plates overnight, and exposed to 10, 25, 50 or 100 μM concentrations of troglitazone or CH in the growth media for 2 hours. Cells were fixed with 2.5% glutaraldehyde in phosphate buffer, harvested, and pelleted. Pellets were post-fixed in 1% osmium tetroxide, and processed to epoxy resin.

Oxidative stress and inducible nitric oxide synthase (iNOS) have been implicated in endothelial cell dysfunction in diabetic retinopathy by formation of peroxynitrite, a cytotoxic product of superoxide and high levels of nitric oxide. Spontaneous diabetic models do not allow for separating the oxidative stress as a result of hyperglycemia from that of the nitric oxide system. in addition chemicals such as streptozotocin which can induce diabetes in animal models cause oxidative stress and induce iNOS before the onset of hyperglycemia. Galactose has been used to induce non-insulin dependent diabetes in mice and rats while gene knockout mice provide a means to study the role of specific NOS components. This study used cytochemical localization of hydrogen peroxide produced by NADH oxidase, a marker for superoxide production, and immunocytochemical localization of nitrotyrosine, a marker for nitric oxide formed radical peroxynitrite, to investigate the role of oxidative stress and the nitric oxide system in galactose induced diabetic retinopathy in iNOS knockout (iNOS%) mice.

Although nuclear channels (NC) have been described in a variety of cells, their function has not been fully investigated. This investigation used immunofluorescent techniques to examine the formation and function of NC in HeLa cells, a human cervical cancer cell line. NC are defined as invaginations of the nuclear envelope (NE) that traverse the nucleoplasm, thus forming a ring-like nucleus. The resulting tunnel is devoid of nucleic acid and may contain cytoplasmic elements. to date, all the reports of the presence of NC are from electron microscopic investigations, which clearly demonstrate the occurrence of NC in the presence of what appear to be interphase nuclei. This investigation was initiated because the NC that we observed in HeLa cells could be seen with fluorescent microscopy and only appeared during telophase and early interphase.

HeLa cells, obtained from American Type Culture Collection, were cultured on glass coverslips in minimum essential medium (MEM) containing 5% calf serum (CS) and a mixture of 5% CO2 in air at 37°C.

The levels of plasma high density lipoproteins (HDL) are inversely related to the incidence of atherosclerosis and coronary heart disease. This protective effect of HDL is thought to be involved in the reverse transport of cholesterol from peripheral tissue to the liver. The scavenger receptor class B type 1 (SR-BI) is the first HDL receptor that can mediate the selective uptake of HDL cholesterol in steroidogenic tissues and in liver. The expression of SR-BI in liver appears to be responsive to changes in the hormonal status and in the levels of HDL cholesterol. However, long-term effects of high-cholesterol diet on the SR-BI expression in liver parenchymal cells and KupfFer cells have not been studied. to investigate the in vivo effects of liver-specific SR-BI expression on lipid metabolism, we fed male Sprague-Dawley rats with high-cholesterol or normal diet for 1 and 12 months.

It is well-known that high-density lipoprotein (HDL) mediates the transport of cholesterol from peripheral tissues to liver through “reverse cholesterol transport” for metabolism. However there is considerable debate about the mechanisms by which HDL removes excess cholesterol from cells. Two different pathways have been suggested: (i) a docking receptor promots cholesterol translocation, or (ii) a receptor mediates intracellular endosomal pathway termed “retroendocytosis“. Our previous studies have indicated that the removal of cholesterol from aortic endothelial and smooth muscle cells in the presence of HDL is facilitated by plasmalemmal vesicles. in this study, our emphasis is on the ultrastructural changes of cholesterol-loaded endothelial cells after incubated with HDL at different time intervals. Furthermore, the mechanism of HDL-mediated cholesterol efflux by cultured aortic endothelial cells is investigated by immunoelectrophoresis.

Confluent monolayers of endothelial cells were incubated in cholesterol DMEM medium for 48 hr.

Chronic hyperglycemia is a common feature of all forms of diabetes mellitus and may contribute greatly to the increase in the incidence of atherosclerosis. It has been demonstrated that increasing arterial permeability to the macromolecules can cause low-density lipoproteins (LDLs) to infiltrate and accumulate in the intima. This event is crucial for developing the plaques in the arterial wall. in this study, we use Evans blue to study the permeability of arterial endothelium in the streptozotocin (STZ)-induced hyperglycemic rats. We also use colloidal gold-labeled LDL to investigate in situ the distribution of LDL in the aortic intima of hyperglycemic rats.

The hyperglycemia was induced in male Sprague-Dawley rats (7 weeks) by a single intraperitoneal injection of streptozotocin (60 mg/kg). The blood glucose concentration and hemoglobin glycation were measured once in every week. The uptake of drinking water is monitored in the regular time interval. After 3 months into the experiment, ten hyperglycemic and control rats were anesthetized.

Mechanical stimuli frequently trigger signals leading to increase gene expression, protein synthesis, or mitogenesis. Cardiac fibroblasts respond to changes in ventricular hemodynamic loading by increasing matrix production in models of pressure-overload hypertrophy and myocardial infarction. Recent studies have identified some of the intracellular signaling pathways that mediate the biological effects observed upon mechanical stimulation in vitro. These include mitogenactivated protein kinases (MAPKs) and protein kinase C (PKC) family. MAPKs include extracellular signal-regulated kinase (ERK), C-Jun NH2-terminal kinase (JNK), and p38 MAPK. ERK pathway is frequently stimulated in response to mitogen.

Previous studies demonstrated that mechanical stretch stimulated MAPK activities in normal adult rat cardiac fibroblasts. On the other hand, mechanical stretch also increases transcription and production of the matrix proteins collagen (type I and HI) and fibronectin in cardiac fibroblasts. MAPKs are activated by mechanical stretch during cardiac myocytes hypertrophy through both PKCdependent and independent pathways.

Fumagillin, an antibiotic isolated from the fungus Aspergillus famigatus and first utilized in the 1950’s for its antiprotozoal and antiamoebic properties, has recently been discovered to be a potent inhibitor of blood vessel growth, termed angiogenesis. A previous study indicated that fumagillin was toxic to murine erythroleukemic cells (MEL BB-88) and HeLa cervical carcinoma cells. This study was initiated to understand what effects the chemical has on the cell surface ultrastructural morphology of these two cancer cell lines.

MEL BB-88 cells were treated with 0.0, 5.0, or 10.0 μg/ml fumagillin for 24, 48, 72, and 96 hours, while HeLa cells received 0.0, 1.0, or 5.0 μg/ml fumagillin for the same time periods. Cells were fixed in 2% glutaraldehyde for two hours, postfixed in 1% osmium tetroxide for one hour, dehydrated in an ascending alcohol series, critically point dried, coated with gold palladium, and observed under an ISI Super III scanning electron microscope

Background: Lidocaine is a local anesthetic that is frequently used for spinal anesthesia. in clinical and animal studies, lidocaine is associated with an increased risk of spinal neurotoxicity following spinal anesthesia. The mechanism of lidocaine neurotoxicity has not been fully elucidated, but it does not involve blockade of sodium channels, the mechanism by which lidocaine causes local anesthesia. Previous in vitro studies assessing cytoplasmic calcium, nerve conduction and action potential generation, and cell death, have demonstrated overt neurotoxicity at higher concentrations of lidocaine (≥ 2% [74 mM] of the hydrochloride formulation), but equivocal results at lower concentrations. Clinically available preparations of lidocaine for spinal anesthesia contain 5% or 2.5% lidocaine hydrochloride. in most cases, lidocaine mixes rapidly with cerebrospinal fluid after injection for spinal anesthesia, so that lower concentrations are more clinically relevant. We have therefore evaluated morphologic assessment of plasma membrane blebbing as a more sensitive assay of neuronal injury by lidocaine.

Sulfur mustard [bis-(2-chloroethyl)sulfide; HD] causes incapacitating injuries to the eyes, respiratory tract, and skin. Despite decades of research, the mechanism of toxic action of HD is still poorly understood. One proposed toxicological mechanism that triggers cell death involves DNA damage induced by HD alkylation. According to this hypothesis, HD-induced DNA damage activates NAD+, requiring the enzyme poly(ADP-ribose) polymerase (PARP) to initiate DNA repair processes. Overactivation of PARP leads to depletion of NAD+, and, in efforts to resynthesize NAD+, cellular ATP is depleted, and the cell dies from intracellular energy. However, recent studies only partial support this hypothesis. Niacinamide, a substrate for NAD+ synthesis, prevented NAD+ depletion, but did not significantly prevent cell death following HD exposure. These studies suggest cell death occurs independently of depletion of cellular NAD+ with some other pathogenic processes operating in cell death following HD cytotoxicity.

The present study examined the role of apoptosis in basal cell degeneration caused by HD exposure in the hairless guinea pig.

Intraspinal cyst formed of tissue proper to the tracheobronchial tract has been referred to as bronchogenic cyst. Six cell types of the cyst epithelium have been identified, i.e., ciliated cells, non-ciliated cells, goblet cells, basal cells, Kulchitsky's cells and undifferentiated cells. This report described the detailed morphological alterations of cilia of the lining epithelium of three cases of intraspinal bronchogenic cyst and compared the findings with the ciliary abnormalities of the tracheobronchial epithelium.

Ultrastructural abnormalities of cilia of the intraspinal bronchogenic cyst could be classified into five categories:

1. (A) Cilia showing addition, deletion or disorganization of axonemal minotubules.

2. (B) Swollen cilia containing a single axoneme in an excess amount of matrix: (a) normal 9 + 2 axoneme,(b) stretched axoneme bending toward one side, (c) abnormal axonemal pattern.

3. (C) Compound cilia with multiple axonemal microtubules ensheathed by a common ciliary membrane: (a) two or more normal 9 + 2 axonemes, (b) two or more normal 9 + 2 axonemes sharing doublets, (c) giant compound cilia with numerous normal and abnormal axoneme in random orientation, (d) compound cilia containing cytoplasmic organelles, (e) compound cilia with excessive matrix.

Postischemic lesion following reperfusion have been investigated in the gerbil by temporary occlusion of a common carotid artery. Two phenomena have been reported in injured ischemic cells: the inability to restore mitochondrial function and evidence of plasma membrane damage. For Further understanding the pathophysiological mechanism of ischemic lesion, we have evaluated the various ischemic period and early postischemic reperfusion. in addition to the morphological changes of the dentate gyrus by light microscopy, we employed the immunocytochemical staining for iNOS and eNOS.

The mongolian gerbil, Meriones unguiculatus, were anesthetized with α-chloralose (350mg/kg), bilateral common carotid artery occlusion was induced for 90 minutes. The right common carotid artery was ligated with 5-0 silk suture, and the left was clamped by a micro vascular/miniature aneurysmal clip. After an ischemic period of 90 minutes, restoration of the blood flow of the left common carotid artery was accomplished by withdrawing the microvascular clip.

Parkinson's disease (PD) is characterized by rigidity, tremor, bradykinesia, and postural instability. The proximate cause of these symptoms is striatal dopamine (DA) insufficiency. The motor symptoms of PD can be alleviated by DA replacement therapy. However, late in the course of the disease patients appear to become less responsive to DA replacement. This therapeutic change suggests the possibility of structural and/or functional defects in striatal medium spiny neurons, which receive convergent DA and cortical (glutamate) inputs.

To understand the neuronal reorganization occurring in Parkinson's disease, we used ultrastructural methods to examine the striatum of rats with striatal dopaminergic deafferentation induced by unilateral intranigral injection of 6-hydroxydopamine. After a six month survival, rats were deeply anesthetized with pentobarbital and perfused with 4% paraformaldehyde-1 % glutaraldehdyde solution in 0.1M Sorenson's phosphate buffer (pH 7.4). The brains were removed, post-fixed for 12 hours, embedded in paraffin, and coronal sections cut through the striatum and midbrain.

The cerebellar granule cells of hamsters, mice, primates and human have been examined to study their organisation, outer and inner surfaces, pattern of connectivity on granule cell groups and synaptic relationships with afferent mossy and climbing fibers in the granule cell layer and with Purkinje cell dendritic spines in the molecular layer.

Confocal laser scanning microscopy of hamster cerebellum cortex, using FM4-64 as an intracellular stain, showed in z-series of stacks of optodigital sections, the presence of granule cell groups formed by small, medium and large granule cells, ranging from 3-10 μm in diameter, surrounded by large Golgi cells. The granule cell/Golgi cell ratio was 50 to 4 (Fig. 1).

Cryofractured mouse cerebellar cortex showed the inner organization of granule cell nuclear and cytoplasmic compartments in samples examined with FEURSEM and coated with chromium (Fig. 2), which were compared with corresponding freeze-etching replica images.

One of Prof. Castaing’s most important gifts to the field of X-ray microanalysis was his insight that quantification could only effectively be carried out by procedures which relied on measurements relative to a suitable standard, because in this way is was the functional variation, rather than the absolute value, of most quantities that had to be known or approximated. At the time this was seen to be a necessary step because the state of knowledge about the fundamental parameters of electron solid interactions was regarded as poor. in the intervening fifty years this situation has not changed very much. Even more unfortunately the kinds of problems to which electron beam microanalysis is now applied - such as the analysis of highly inhomogeneous or very small samples - are of such a nature that quantitative methods based on standardization are no longer feasible.

Mechanisms in electrostatic charging of insulating materials during electron irradiation are very difficult to predict because they involve cross-correlated parameters: the surface potential, Vs and the secondary electron yield,δ, for bare specimens; the possible change of the bulk composition induced by the internal electric field for ground coated specimens. A better understanding of these mechanisms requires efforts from both the theoretical and the experimental points of view.

From the theoretical point of view, a two-layer model for distribution of the trapped charges (Q+ over the escape depth of the secondaries, s; Q‐ over the maximum penetration depth R of the incident electrons) often permits to account for many charging effects observed on bare specimens. For instance, the failure of the total yield approach may be partly explained by the fact that the charge distribution expected at the critical energy Ec2 (where δ+ η=l) corresponds to Q+=‐Q‐ but also to R ≫ s at the early beginning of the SEM and EDS investigation.