In the past five years, a case for oxidative stress in the pathogenesis of Alzheimer disease (AD) has been convincingly made. That link owes most of its existence to the microscopic detection of neuronal oxidative damage. Focusing on damage not only has the advantage that stable products are studied, rather than short-lived radicals, but also that damage can be morphologically defined. This latter aspect is critical since biochemical analysis of whole tissue is mostly of the accumulated damage from normal metabolism with aging of long-lived polymers in the extracellular matrix (Figure 1). The importance of damage is that it is the result most likely to be linked to pathology. Driven by the hypothesis that oxidative damage plays a role in the aggregation of insoluble protein in the lesions of Alzheimer disease, neurofibrillary tangles and senile plaques, we developed reagents to detect protein modifications related to glycoxidation, lipid peroxidation, peroxynitrite, free carbonyls and carbonyl-modification (Figure 2).

Proteins of the axonal cytoskeleton, particularly neurofilament and microtubule-associated protein τ, should be particularly sensitive to the effects of oxidative modification due to their high content of lysine, an amino acid that is particularly susceptible to direct oxidization as well as adduction by carbonyls produced from lipid and sugar oxidation. To understand the susceptibility of the cytoskeleton to oxidative modification and whether such modification is related to the physiological function of the cytoskeleton, we undertook a cytological analysis of motor neurons isolated from mouse spinal cord. These neurons contain an abundant axonal cytoskeleton that can be readily analyzed distinct from the cell body. Immunocytochemistry, using antibodies against protein-adducts of the highly reactive lipid peroxidation product, hydroxynonenal (HNE), representing Michael addition or pyrrole formation, revealed that HNE-immunoreactive adducts are found in all axons. This in situ distribution of HNE-adducts is consistent with immunoblots prepared from axons which show selective HNE modification of neurofilament heavy subunit (NFH) but not of other cytoskeletal proteins.

Proteasomes are multicatalytic structures found in cells that play a major role in the proteolytic degradation of proteins. Before entering the proteasome, proteins are marked like trees to be cut by attachment of ubiquitin. Neurodegenerative disorders of aging, including Alzheimer's, show abnormal accumulation of plaques and neurofibrillary tangles (NT). The latter are composed of paired helical filaments containing ubiquitinated tau protein. The fact that ubiquitinated tau is not degraded by the proteasomes may be a key to the formation of NT. Proteasomes, like other cellular pathways, are controlled by various mechanisms. It was found that amyloid β protein (Aβ) inhibits chymotrypsin-like proteasomal activity. Additionally, Aβ was found in elevated quantity in NTs in Alzheimer's diseased brain tissue. Understanding the mechanism of action of Aβ therefore becomes important.

In order to track the regulatory Aβ protein (Aβ1-40), a variant was synthesized with a terminal cysteine (Aβ1-40C40) that could be gold labeled.

Neurofibrillary tangles (NFT) occur with high frequency in brains of patients with Alzheimer' disease (AD). The frequency of NFT directly correlates with the degree of dementia. Extra cellular plaque core amyloid also occurs in high frequently in AD brains when few intraneuronal NFT are present, these individuals are not demented.

The microtubule associated protein (MAP) tau is recognized as the principal constituent in paired helical filaments (PHF) and NFT. NFT contain PHF, amorphous structures containing tau, and a triple-stranded left-hand helical ˜2.1 nm filament similar to tau polymer which has been identified in isolated tangles. Tau is located in nerve cell axons and associates with and stabilizes the microtubules required for axonal transport. Sequestration of tau into tangles and its removal from microtubules not only compromises axonal transport but cripples synaptic communication between nerve cells.

NFT were isolated in the absence of detergent so these gel-like structures (Fig. 1) could be studied at the molecular level.

Although there are a variety of gasoline ethanol mixtures proposed as neat fuels (ethanol 85% + gasoline 15% = E85; E95) for automobiles, gasohol (gasoline 90% + ethanol 10%) is presently used as a fuel in the United States. The adverse effects, if any, of gasohol ingestion are unknown; effects on the liver of rats administered gasohol are examined in this study.

Twenty-four female Sprague-Dawley rats received daily, via gavage, one of the three concentrations of gasohol for 28 days; LD50/20, LD50/100 and LD50/1000, where LD50 = 1.5g ethanol / kg body weight (bw) and 14g gasoline / kg bw. The LD50 was based on that of gasoline, which was obtained from literature value.1 The amount of ethanol added to stock gasohol was only 1/10 its LD50, required to maintain the gasoline ethanol proportion of 9:1. Gasohol was administered in corn oil with total volume 10 ml. Animals that received only corn oil served as controls.

Polyhalogenated aromatic compounds such as polychlorinated biphenyls (PCBs) and polychlorinated dibenzo-p-dioxins (PCDDs) continue to be environmental contaminants because of their bioaccumulation in the food chain and high resistance to biodegradation. The current study was undertaken to determine if a mixture of PCB congeners (WHO-IPCS) were interactive with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in producing morphological changes in the rat liver. Both compounds are known to produce a broad range of biochemical and morphological alterations including enzyme induction.

Groups (N=5) of female Sprague-Dawley rats were administered TCDD (0, 2.5, 25, 250, 1000 ng/kg bw/day) or PCB (0, 2, 20 μg/kg bw/day) alone, or in combination with each concentration of both compounds. Incorrect concentrations were published in a previous abstract. The test substance was mixed with corn oil and given by gavage at 2 ml/kg daily for 28 days. At the end of the experiment, the rats were killed and liver samples were prepared for transmission electron microscopy.

Herbicides and insecticides are applied in agriculture sequentially or simultaneously to control broadleaf weeds, corn rootworm larvae, and other soil pests. Pesticide application creates the potential for movement to aquatic habitats via sediment or runoff, hence affects non-target aquatic organisms. Histopathological changes of gill tissues have been suggested as powerful and sensitive measures of stress and well-being offish. Atrazine and terbufos were found to cause tissue damage and mortality in fish. Literature concerning the effects of pesticide mixtures to fish and other animals at the histopathological level are rare. The present study focused on the impacts of atrazine and terbufos mixtures on fish gills.

Cyprinella lutrensis were obtained from the Platte River in Nebraska, USA and maintained in the laboratory at 23 °C and 30 °C. They were exposed to 0, 1μg terbufos + 10 μg atrazine L-1, 10 μg terbufos + 100 μg atrazine L-1, and 100 μg terbufos + 1000 μg atrazine L-1.

Although liver function tests could be abnormal in humans taking cocaine the histopathological basis for this disorder has not been well established. Light microscopic studies have shown the existence of peripheral, centrilobular or diffuse necrosis. The only electron microscopic investigation we could find reports hepatocyte alterations including dilated rough endoplasmic reticulum, hypertrophy of smooth endoplasmic reticulum, and existence of phagolysosomes. In this work we report the liver ultrastructural pathology in chronic cocaine users.

Liver biopsies were obtained in five male patients, 25-44 years old. These patients had consumed cocaine and other drugs (marihuana, alcohol, amphetamines, etc..) for 7-30 years. All of them had altered liver function tests. Tissue samples were processed with routine techniques for transmission electron microscopy and observed in a Hitachi H-500 electron microscope.

Abnormalities observed included those previously reported as swollen rough and smooth endoplasmic reticulum, presence of autophagic vacuoles and lipid deposition.

Poly-(trans-l,2-diaminocyclohexane) platinumj-carboxyamylose (Poly-Plat); 5-SuIfosalicylato-trans-(l,2-diaminocyclohexane) platinum (SSP); and 4-Hydroxy-a-sulfonylphenylacetato (trans 1,2-diaminocyclohexane) platinum (II) (SAP) (Andrulis Pharmaceuticals, Bethesda, MD) are three novel second generation platinum containing antineoplastic compounds. Initial studies indicate that these agents are more effective in the treatment of cancer while at the same time less toxic to the organism as a whole than cisplatin (CDDP). The present study was undertaken to examine the effects of these new compounds on glucose-6-phosphate dehydrogenase (G-6-PDH) as compared to CDDP treated and normal kidney and liver tissues.

Wistar rats (100-120g) were given intraperitoneal injections of CDDP (9 mg/ kg) and Poly-Plat, SSP and SAP (10 mg/ kg) over a 5 day period. On day 6 the animals were sacrificed and tissues (kidney and liver) were freeze sectioned (7 μm). Sections were incubated in media according to the accepted method specific for the G-6-PDH localization at a pH of 7.46 for 30 min.

Redox dysregulation is known as pathogenesis of renal involvement in autoimmune lupus mice [1,2]. However, the behavior of reactive oxygen species (ROS) is still unclear [3]. The purpose of this study is to make clear the localization of hydrogen perhydroxide which is generated as intermediate substances of radical reactions.

Modified Brigg's method [4] was used to determine the generation of ROS in renal tissues of (NZB x NZW)F1 mice. The renal specimens were incubated with a standard medium, which consisted of 0. IM tris-malate buffer (pH7.4) with 7% sucrose, lmM CeCl3 and lOmM aminotriazole at 37° C for 30 min and ultrathin sections were prepared and examined by electron microscopy. Lecithinized-recombinant-superoxide dismutase (SOD) was used as redox regulator, which is ROS inhibitor. SOD (1000 U/kg) was injected 3 times a week, tail-intravenously to Fl mice from 4 to 60 weeks of age. The mRNA of inflammatory cytokines and IkB was detected by RT-PCR method.

When confluent monolayers of Madin-Darby Canine Kidney (MDCK) renal epithelial cells are exposed to calcium oxalate monohydrate (COM) crystals suspended in the nutrient media, crystals settle onto the cell surface. Here, they become entangled with the specialized membrane structures of cilia and microvilli (Figures 2). The cell membrane surface is structurally altered as patches of membrane devoid of microvilli are found adjacent to the crystal entrapped on the surface (Figure 4). Many COM crystals disappear from the cell surface and appear to be internalized. This phenomenon is seemingly common to all cultured renal epithelial cells. The uptake of crystals appear to increase with time and crystal concentration and stimulates a series of complex reactions. Morphologically, crystal aggregates are contained with intracellular vesicles which appear to be membrane lined (Figure 2,3). In addition, cells exocytose the internalized crystals and in MDCK cells, crystal-cytoplasm complexes can be observed leaving cells at the intercellular junctions.

Acute myocarditis is a potentially lethal disease. However, the precise mechanism of myocardial damage in myocarditis is still unknown, although its pathogenesis seems to be involved in the result of natural killer cells, cytotoxic T cells and autoantibodies. Recently, it has been reported that programmed death of cardiac myocytes can be induced by several pathological conditions including infarction and end-stage cardiac failure. However, little is known about the myocardial cell death in myocarditis. In this study, we investigated whether myocardial cell death via apoptosis occurs in the heart tissue with myocarditis.

Since it has been reported that the pathogenesis of the tissue damage in viral myocarditis resembles that in experimental autoimmune myocarditis induced by myosin, we used an experimental model for autoimmune myocarditis induced in Lewis rat with the use of myosin as the antigen. Rats were sacrificed on days 14, 17 and 21 after myosin injection. The tissue specimens were taken from ventricle of the heart.

Sulfur mustard gas (HD), a synthetic vesicating agent used effectively as a major chemical warfare agent during World War 1, continues to be a modern day threat agent. Unfortunately there is no specific pretreatment or antidotal therapy for those who may become exposed. Whole body exposure results in cutaneous, respiratory and ocular effects. of these, eye impairment leads to the most immediate incapacitation. However HD-induced eye lesions remain to be fully characterized. In the present study we explore histological, ultrastructural and immunopathological effects of a vesicating dose of HD in rabbit cornea occurring during the first 24 hours following exposure.

A 0.4μl drop of liquid HD was placed on the left cornea of anesthetized rabbits. The right cornea served as an unexposed control. Following exposure animals were returned to their cages and given appropriate care by an attending veterinarian. Eye injury was evaluated by clinical observations and given scores of severity from simple conjunctival redness to apparent corneal damage.

Cationic amphiphilic drugs (CAD) are structurally characterized by their hydrophobic ring structure and hydrophilic side chain. While there is little known about the effect of CAD in dogs, studies in animal models have shown that over 30 CAD with various pharmacological activities have the ability to induce phospholipid-like inclusions in peripheral blood cells and in organs such as lung, liver, eye and spleen. Two CAD used for treatment of human disease are amiodarone, prescribed for arrythmia, and chloroquine, an antimalarial. The purpose of this study was to determine if these compounds induce cytoplasmic phospholipid inclusions in peripheral blood mononuclear cells (PBMC), polymorphonuclear lymphocytes (PMN), alveloar macrophages, liver and retina in dogs.

Samples were collected from male beagle dogs after 6 weeks of oral dosing with amiodarone (75 mg/kg/day) or chloroquine (75 mg/kg/day). Buffy coats were prepared from peripheral blood and fixed with 3% glutaraldehyde in 0.1 M phosphate buffer, pH 7.2. Alveolar macrophages were recovered from bronchoalveolar lavage fluid by centrifugation and the pellets fixed with buffered 2.5% glutaraldehyde-2% formaldehyde.

It has been proposed that sphinganine may function as a natural antifungal barrier in mammals. This sphingoid base is found highly concentrated in epidermis, where it has been documented to inhibit the colonization of pathogenic fungi such as Epidermophyton and Candida. Since Neurospora crassa is sensitive to the effects of sphinganine within the same concentration range as infectious fungi, it is being employed as the model to study the mechanism of sphinganine toxicity in fungal pathogens. With the dramatic increase in fungal mycoses seen in immunocompromised individuals to date, this natural constituent of mammalian epidermis may be employed as a potent, novel antifungal drug.

During this project, radioactive labelling with [3H]sphinganine was employed to observe metabolism of exogenous sphinganine into complex sphingolipids. Transmission electron microscopy analysis was utilized to observe changes in cellular ultrastructure, while fluorescence microscopic techniques were used to assay certain cell functions.

Within 1 hr, exogenous sources of sphinganine are metabolized into dihydroceramides and cerebrosides by the enzymes of complex sphingolipid synthesis.

The incidence of bladder stones is increased by 50% following bladder augmentation(l,2). Stones require surgical removal as they are typically too large to pass. A recurrence rate of 19% has been noted(2) necessitating multiple sugical procedures. Etiology of stones in augmented bladders is due to a number of factors including foreign body reactions, metabolic abnormalities, infection and intestinal mucus. In order to determine whether the elemental composition of bladder mucus plays a role in the development of bladder calculi, energy dispersive x-ray spectrometry(EDXS) was done on the mucus from stone and non-stone forming patients. In addition, stones from the same patients were examined and analyzed and the data compared with the composition of the mucus.

Eighteen patients participated in this study, 14 of whom had ileo-cystoplasties and 2 ileo-cecal augments. Two were cloacal extrophy patients where the hind gut was incorporated with the bladder halves. To avoid any foreign body reactions, metal staples were not used in any of the bladder augmentation procedures.

Prolonged bleeding times may be evident in patients with various conditions. Analysis of platelet function can provide an insight into the cause. Platelets contain substances such as ADP, calcium, epinephrine, and serotonin which are stored in the electron dense or delta granules. Our experience has shown normal platelets to have 4-6 dense granules (Figure 1), which is supported by the literature. The prolonged bleeding times of patients who have less than the normal number of dense granules may be attributed to the lack of adequate factors that aid in the clotting process. These storage pool deficient (SPD) patients are being classified by their low dense granule count. Patients with prolonged bleeding times but normal dense granule counts may need to have their granules evaluated for size and volume (Figure 2).

Whole mounted platelets were placed in the transmission electron microscope (TEM), and negatives were obtained at a magnification of 10,200x. The typical dense granule seen in normal whole mounts is circular.

Two fundamental properties of protoplasm: contractility and conductivity, are brought to a high degree of development in muscular tissue. The rhythm of the mammalian heart is initiated and regulated by fibers that have become specialized to conduct the electrical impulse. These fibers comprise the impulse conduction system of the heart, and they are capable of the conduction of an impulse at a faster rate than the rest of the myocardium, whose fibers are specialized for contraction, which has earned them the terminology of “working myocardium”. Conduction tissue has the property of automaticity, which is defined as the ability of a cell to depolarize spontaneously during the diastolic interval and to reach the threshold potential, thus generating an action potential. The conducting cells have a lower internal resistance and a higher conductance than the tension generating cells of the myocardium, which are specialized for contraction. This tissue shows a prepotential rise of the baseline, so that the cells are already partially depolarized.

Ebola and Marburg viruses, members of the family Filoviridae, cause a severe and often fatal hemorrhagic fever. In April and May of 1995, an outbreak of Ebola virus hemorrhagic fever occurred in Kikwit, Zaire, and of 318 known cases, approximately 77% died. The ultrastructural pathology associated with filoviruses has been well described in non-human primates. On the other hand, ultrastructural studies on human tissues have been relatively few, and limited for the most part to examination of the liver. We report here our ultrastructural observations of autopsy tissues from the liver, as well as lung, skin, and testes of infected patients by using both thin section electron microscopy (EM) and immunogold labeling electron microscopy (IEM).

At the hospital in Kikwit, strict barrier precautions were employed to collect tissue specimens. These were diced into small blocks, placed into fixative, and later transferred to phosphate buffer. Specimens were hand-transported to the CDC in Atlanta, gamma-irradiated, and embedded in either Epon-substitute/Araldite for standard EM, or in LR White for IEM.

Molecular and immunologie assays for virus identification and characterization are well known for their high specificity and sensitivity. High specificity occasionally hampers efforts to detect virus strains distantly related to previously recognized types and may allow infectious agents to be undetected when these techniques are used for identification. We describe in this report such an example with an outbreak of foodborne gastroenteritis.

Forty-six cases of gastrointestinal illness were reported in May 1994 among adult staff of a Parkville, Md., school after a catered luncheon. Symptoms included stomach cramps, nausea, diarrhea, and vomiting. Seven stool specimens, negative by routine bacteriological screening, and 14 acute-and convalescent-phase sera from patients involved in the outbreak were tested for viruses. The stool specimens were examined by electron microscopy (EM) and reverse transcription-polymerase chain reaction (RT-PCR) designed to detect small round structured viruses (SRSVs). In addition, serum specimens were tested for IgG antibody to recombinant capsid proteins from Norwalk virus (rNV) and Toronto virus (rTV) using a direct enzyme immunoassay (EIA).