mTOR Modulates Methamphetamine-Induced Toxicity through Cell Clearing Systems.

Methamphetamine (METH) is abused worldwide, and it represents a threat for public health. METH exposure induces a variety of detrimental effects. In fact, METH produces a number of oxidative species, which lead to lipid peroxidation, protein misfolding, and nuclear damage. Cell clearing pathways such as ubiquitin-proteasome (UP) and autophagy (ATG) are involved in METH-induced oxidative damage. Although these pathways were traditionally considered to operate as separate metabolic systems, recent studies demonstrate their interconnection at the functional and biochemical level. Very recently, the convergence between UP and ATG was evidenced within a single organelle named autophagoproteasome (APP), which is suppressed by mTOR activation. In the present research study, the occurrence of APP during METH toxicity was analyzed. In fact, coimmunoprecipitation indicates a binding between LC3 and P20S particles, which also recruit p62 and alpha-synuclein. The amount of METH-induced toxicity correlates with APP levels. Specific markers for ATG and UP, such as LC3 and P20S in the cytosol, and within METH-induced vacuoles, were measured at different doses and time intervals following METH administration either alone or combined with mTOR modulators. Western blotting, coimmunoprecipitation, light microscopy, confocal microscopy, plain transmission electron microscopy, and immunogold staining were used to document the effects of mTOR modulation on METH toxicity and the merging of UP with ATG markers within APPs. METH-induced cell death is prevented by mTOR inhibition, while it is worsened by mTOR activation, which correlates with the amount of autophagoproteasomes. The present data, which apply to METH toxicity, are also relevant to provide a novel insight into cell clearing pathways to counteract several kinds of oxidative damage.

Chronic alcohol administration causes expression of calprotectin and RAGE altering the distribution of zinc ions in mouse testis.

Several studies reported that chronic alcohol consumption alters the intestinal mucosa barrier, and subsequent entrance of endotoxins into the bloodstream. In many tissues endotoxin exposure causes the expression of calprotectin (CP) and the receptor for advanced glycation -end products (RAGE). In this study we investigated whether chronic alcohol administration causes expression of CP and RAGE in mouse testis. The distribution of free and loosely bound Zn(2+) (FLB-Zn(2+)) in the testicular tissues was also evaluated. Alcohol-induced testicular damage was documented by measuring testosterone blood levels and by light and electron microscope studies. Twenty mice were treated daily for three weeks with 3.0 g/kg of a 25% solution of alcohol. Ten mice were treated in the same period of time with a solution of maltose dextrins, isocaloric to alcohol. Twenty untreated mice were used as controls. Alcohol treated mice showed diffuse expression of CP and RAGE in the interstitial cells. RAGE was found also in the basal compartment of the seminiferous tubules. Depletion of FLB-Zn(2+) was observed in the adluminal compartment of the seminiferous tubules. Expression of CP and RAGE was not found in control mice and maltose dextrin treated mice. Our results indicated novel mechanisms by which alcohol acts in testis. Indeed, CP and RAGE may cause the generation of oxidants and inflammatory mediators, with negative impact on testicular functions. Depletion of FLB-Zn(2+) may contribute to the dysregulation of spermatogenesis.

Immunohistochemical localization of 3-nitrotyrosine in the nasal respiratory mucosa of patients with vasomotor rhinitis.

CONCLUSION: This study demonstrates that, in the nasal respiratory mucosa of patients with vasomotor rhinitis, oxidative stress following peroxynitrite formation is confined to the respiratory epithelium. This suggests that the role of peroxynitrite in vasomotor rhinitis differs from its role in other diseases of the respiratory tract. The results of this study also support the concept that different pathogenetic mechanisms are probably involved in vasomotor rhinitis. OBJECTIVE: Previous studies indicated that nitric oxide (NO) is involved in the pathogenesis of vasomotor rhinitis, strong expression of NO synthase being detected in the smooth muscle cells of the cavernous sinuses and in the respiratory epithelium. However, most adverse effects of high levels of NO originate from the reaction of NO with superoxide anions to form peroxynitrite. Therefore, in this study we evaluated the involvement of peroxynitrite in the pathogenesis of vasomotor rhinitis. MATERIAL AND METHODS: Sites of peroxynitrite formation were identified by immunolabelling for 3-nitrotyrosine (3NT), its footprint in tissues. Samples of nasal mucosa were obtained from vasomotor rhinitis patients and from control subjects who had undergone corrective surgery of the nasal septum. All samples were obtained by reduction of the inferior turbinate. RESULTS: Examination of specimens from vasomotor rhinitis patients revealed that 3NT is absent in epithelium with a normal appearance, cells of the subepithelial connective tissue, the glands and the blood vessels, including the cavernous sinuses. In contrast, intense 3NT immunolabelling was found in the disrupted respiratory epithelium. 3NT was not present in any of the specimens from control subjects.

Ultrastructure of testicular macrophages in aging mice.

Testicular macrophages of aging mice were studied by TEM. Testicular macrophages retained with Leydig cells the close morphological relationships observed in the adult young animals, but digitations were not found. Lipofuscin granules like those of the Leydig cells from aging mice were observed in the cytoplasm. These organelles were generally absent in the testicular macrophages of young adult mice. Testicular macrophages did not display phagocytosis of the lipofuscin granules. In addition, the latter were not found in the intercellular spaces. These observations indicated that lipofuscin granules were formed, at least in a great part, within testicular macrophages as a consequence of metabolic changes occurring with age. Fine lamellar organization was seen in the lipofuscin granules of both Leydig cells and testicular macrophages. Frequently, lipofuscin granules originated from secondary lysosomes containing lipidic vacuoles only. Together with accumulation of the lipofuscin granules, changes of testicular macrophage fine morphology were observed. Endoplasmic reticulum and Golgi apparatus became poorly developed, and coated vesicles were rarely found. Fewer mitochondria were encountered, but their ultrastructure was not altered. These results suggest that in testicular macrophages lipofuscin accumulation is associated with a functional involution.

Ultrastructural and ultracytochemical study of the human nasal respiratory epithelium in vasomotor rhinitis.

OBJECTIVES: Several pieces of evidence have suggested that nitric oxide (NO) fulfills important functions in the respiratory mucosa, under both normal and pathological conditions. This study was performed to investigate the role of NO in the nasal respiratory epithelium of patients affected by vasomotor rhinitis. The structure and ultrastructure of the epithelium were also examined. MATERIAL AND METHODS: The localization of NO synthase activity was determined by means of reduced nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase ultracytochemistry. Nasal mucosa was obtained from patients who had undergone surgical therapy for reduction of the inferior turbinate. RESULTS: Examination of hematoxylin-eosin-stained sections revealed that most of the nasal mucosa covering the surgical samples was characterized by severe epithelial damage. The ultrastructural study confirmed the light microscopic observations. Ciliary loss, absence of the intercellular junctions and distension of the intercellular spaces were found in the damaged epithelium. The basement membrane was frequently interrupted. Some epithelial cells were identified as basal cells. Other cells of the damaged epithelium were probably involuted ciliated and goblet cells. The ultracytochemical study showed that the basal cells were NADPH-diaphorase-negative in healthy subjects and strongly NADPH-diaphorase-positive in subjects with vasomotor rhinitis. CONCLUSIONS: It is suggested that NO has cytotoxic effects and causes inhibition of mitotic activity in the basal cells, leading to epithelial disruption and breakdown of the protective functions of the epithelium.

ETA receptor-mediated Ca2+ mobilisation in H9c2 cardiac cells.

Expression and pharmacological properties of endothelin receptors (ETRs) were investigated in H9c2 cardiomyoblasts. The mechanism of receptor-mediated modulation of intracellular Ca(2+) concentration ([Ca(2+)](i)) was examined by measuring fluorescence increase of Fluo-3-loaded cells with flow cytometry. Binding assays showed that [125I]endothelin-1 (ET-1) bound to a single class of high affinity binding sites in cardiomyoblast membranes. Endothelin-3 (ET-3) displaced bound [125I]ET-1 in a biphasic manner, in contrast to an ET(B)-selective agonist, IRL-1620, that was ineffective. The ET(B)-selective antagonist, BQ-788, inhibited [125I]ET-1 binding in a monophasic manner and with low potency. An ET(A)-selective antagonist, BQ-123, competed [125I]ET-1 binding in a monophasic manner. This antagonist was found to be 13-fold more potent than BQ-788. Immunoblotting analysis using anti-ET(A) and -ET(B) antibodies confirmed a predominant expression of the ET(A) receptor. ET-1 induced a concentration-dependent increase of Fluo-3 fluorescence in cardiomyoblasts resuspended in buffer containing 1mM CaCl(2). Treatment of cells with antagonists, PD-145065 and BQ-123, or a phospholipase C-beta inhibitor, U-73122, abolished ET-1-mediated increases in fluorescence. The close structural analogue of U-73122, U-73343, caused a minimal effect on the concentration-response curve of ET-1. ET-3 produced no major increase of Fluo-3 fluorescence. Removal of extracellular Ca(2+) resulted in a shift to the right of the ET-1 concentration-response curve. Both the L-type voltage-operated Ca(2+) channel blocker, nifedipine, and the ryanodine receptor inhibitor, dantrolene, reduced the efficacy of ET-1. Two protein kinase C inhibitors reduced both potency and efficacy of ET-1. Our results demonstrate that ET(A) receptors are expressed and functionally coupled to rise of [Ca(2+)](i) in H9c2 cardiomyoblasts. ET-1-induced [Ca(2+)](i) increase is triggered by Ca(2+) release from intracellular inositol 1,4,5-trisphosphate-gated stores; plasma membrane Ca(2+) channels and ryanodine receptors participate in sustaining the Ca(2+) response. Regulation of channel opening by protein kinase C is also involved in the process of [Ca(2+)](i) increase.

Ultrastructural and ultracytochemical study of the middle ear epithelium in the chicken, Gallus gallus domesticus.

The fine structure of the epithelium lining the tympanic cavity of the chicken was studied by TEM and SEM. In addition, the distribution of nonspecific esterase activity in the epithelium was investigated by TEM. Ultrastructural study revealed the presence of disk-like apical protrusions of the epithelial cells, previously not observed in other cell types. The protrusions contained some cytoplasmic organelles and were characterized by a ring-shaped thickening around their periphery. The ring was made up of a granulo-filamentous material. Our observations clearly indicate the existence of an apocrine secretory mechanism, consisting of a progressive detachment of disk-like protrusions from the apex of the epithelial cells. The ultracytochemical study demonstrated nonspecific esterase activity on the epithelial surface and in the secretory vesicles. We propose that nonspecific esterase is a marker for middle ear surfactant in birds.

Endothelial cell binding by systemic lupus antibodies: functional properties and relationship with anti-DNA activity.

Anti-DNA antibodies and anti-endothelial cell antibodies (AECA) are often detected in systemic lupus erythematosus (SLE). Anti-DNA antibodies can also bind the membrane of human umbilical vein endothelial cells (HUVEC), but little is known about the presence of AECA in the population of immunoglobulins from SLE sera that do not bind DNA. The aim of this study is to analyse the ability of anti-DNA and non-anti-DNA antibodies from SLE sera to bind endothelial cell antigens and to investigate their pathogenic potential. Both anti-DNA and non-anti-DNA antibodies display AECA activity by immunoprecipitation and flow cytometry and in some patients recognize antigens of identical molecular weight. Complement-dependent cytotoxicity on HUVEC was not detected with either anti-DNA or non-anti-DNA antibodies. Similarly, apoptosis was not induced in HUVEC and HL60 incubated with anti-DNA or non-anti-DNA antibodies, as shown by the DNA hypodiploid content. These data indicate that AECA are highly heterogeneous, as they recognize a wide variety of surface molecules on HUVEC and equally present in anti-DNA and non-anti-DNA antibodies from SLE patients.