Cellular and subcellular distribution of metals in molluscs

The cellular processes involved in metal metabolism in molluscs are reviewed, with emphasis on the contribution of microscopy (AMG, ARG, EPMA, and SIMS) to both basic research of metal cell biology and applied environmental research. In molluscs, metal uptake may occur by facilitated diffusion, active transport, or endocytosis, and can be enhanced by MT synthesis or formation of mineralized granules. In aquatic molluscs, gills constitute a key interface for dissolved metal uptake, where metals are bound to MT, incorporated into lysosomes, and released basally towards the blood plasma and circulating hemocytes. However, particulate metal uptake is mainly achieved via the digestive tract by endocytosis; further metals are transferred first to lysosomes and then to residual bodies, especially in the digestive cells of the digestive gland. Additionally, metals can be accumulated selectively in specific cell types. As ligands pools differ from cell to cell, different metals may be retained in different cell types. Class "a" metals are localized in cells with granules composed of carbonate, oxalate, phosphate, and sulfate (oxygen donors), whereas "b" metals are associated with those cell types rich in sulfur and nitrogen ligands (sulfur donors). In molluscs, oxygen donors occur in connective tissue calcium cells and basophilic cells, whereas sulfur donors are present in digestive cells, podocytes, nephrocytes, and rhogocytes. Hemocytes, which constitute the most relevant system for metal transport between tissues, move around the body and may penetrate tissues and remove metals from the inner medium to be accumulated in lysosomes as nondigested products. Rhogocytes also participate in metal mobilization, accumulation, and release. The assessment of metal levels in target cells of sentinel molluscs by microscopic techniques provides an early-warning measure, with promising applications as an exposure biomarker for environmental monitoring programs.     

Age-related changes of NADPH-diaphorase-positive neurons in the rat inferior colliculus and auditory cortex

Nitric oxide (NO) has been implied in age-related changes of the central nervous system (CNS) and the central auditory pathway. The present study was conducted to investigate whether the number of NO-producing cells and their morphometric characteristics in the inferior colliculus (IC) and the auditory cortex (AC) are changed with the increasing age of the subjects. IC and AC sections of adult and senile Wistar rats were studied using the histochemical detection of NADPH-diaphorase activity (NADPH-d), a marker for neurons containing nitric oxide synthase (NOS). Our results showed a decreased area of the somas of NADPH-d-positive neurons in the dorsal cortex (DC) of the IC and a diffuse loss of NADPH-d-positive neurons in the senile IC and primary cortical auditory area (Te1). However, an increased number of NO-producing cells have been shown by other authors in different parts of the ageing auditory pathway and CNS. It seems that age-related changes in NADPH-d-positive cells may follow a region-specific route. These changes may be related to hearing impairments with increasing age.     

Neuronal nitric oxide synthase immunoreactive neurons in the mammalian retina

The development of immunocytochemistry has led to a better understanding of synaptic transmission carried out by neuroactive substances in the mammalian brain, including the retina. In the mammalian retina, nitric oxide (NO) is widely accepted as a neuromodulator. Histochemistry based on NADPH-d and immunocytochemistry based on nitric oxide synthase (NOS) have been used to identify the presence of nitric oxide in the mammalian retina. Certain types of amacrine cells and a class of displaced amacrine cells have been labeled consistently in all mammalian retinae studied to date. Other cell types showing NADPH-d reactivity or NOS immunoreactivity varied between species. NADPH-d reactive or NOS immunoreactive amacrine cells may serve as a source of NO for amacrine, bipolar, and ganglion cells in the inner retina, whereas interplexiform cells, bipolar cells, and horizontal cells may serve as a source of NO for the outer retina of mammals.     

Structure and secretory activity of cultured chondrocytes from patients with osteoarthritis.

Cartilage samples were taken from OA patients in order to describe and quantify pro-inflammatory mediators. Samples were cultured under aseptic conditions in Dulbecco's modified Eagle medium at 37 degrees C for 10 days. Control samples, taken from non-inflammatory cartilage, were cultured under the same conditions. The levels of NO(-)2 and NO(-)3 were measured in the supernatant using a spectrophotometric assay. The activity of MMP-1 was quantified by ELISA. The concentration of NO(-)x was 47.3 +/- 4.1 microM in the OA cartilague and 10.7 +/- 1.8 microM in the controls. The average MMP-1 activity was 3,650 +/- 387 ng/ml in the OA cartilage and 2,150 +/- 190 ng/ml in the control samples. These increased values of MMP-1 and NO(-)x observed in the OA cartilage suggest a higher catabolic activity. A morphological analysis of OA chondral tissue using light microscopy shows that the surface of the tissue is characterized by the presence of aggregated chondrocytes or "clones" but in the deeper areas isolated cells are found. These results could be a significant contribution towards the identification of biological markers indicating the presence of OA activity.     

Cyclic GMP signaling in podocytes

Natriuretic peptides (NP), together with nitric oxide (NO) are powerful relaxing factors acting via a common second messenger, cyclic GMP (cGMP). Together with other vasoactive modulators, these vasorelaxing factors play an essential role in regulating the function of kidney glomeruli. The presence of NP receptors in podocytes has been well documented. Recently, also mRNA for soluble guanylate cyclase, the NO receptor, has been shown in these cells. Stimulation of podocytes with atrial natriuretic peptide (ANP), C-type natriuretic peptide (CNP), and NO donors results in considerable upregulation of cellular cGMP synthesis. The podocyte foot processes contain a highly organized network of microfilaments adhering to the glomerular basement membrane (GBM). Changes in podocyte cytoskeleton accompanied by detachment of the cells from the GBM are closely associated with many glomerulopathies. The contractile apparatus in the podocyte foot processes seems to be an obvious target for the cyclic GMP signaling cascade. However, little is known about implications of the cGMP synthesis in these cells. We briefly review the current art regarding generation and modulation of cyclic GMP levels in podocytes. We discuss also the possible targets for this secondary messenger as well as its functional role in podocytes.     

Fine structure of olfactory epithelia of gastropod molluscs.

Among gastropod molluscs the chemical senses are most important for location of distant objects. They are used in food finding, locating mates, avoiding predators, trail following, and homing. Chemoreceptors are commonly associated with the oral area, the tentacles, and the osphradium, which lies in the mantle cavity. Most chemosensory neurons are primary sensory neurons, although secondary sensory cells have been reported in the osphradium of some prosobranch gastropods. Most chemosensory organs contain sensory cells with ciliated sensory endings that are in contact with the external environment. Some sensory endings have only microvilli or have no surface elaborations. Cilia on sensory endings are commonly of the conventional type, but some species have modified cilia; some lack rootlets, some have an abnormal microtubular content, and some have paddle-shaped endings. The perikarya of sensory neurons may be within the sensory epithelium, below it, or in ganglia near the sensory surface. In some groups of gastropods there are peripheral ganglia in the olfactory pathway; in others chemosensory axons appear to pass directly to the CNS. Olfactory epithelia of terrestrial pulmonates have modified brush borders with long branching plasmatic processes and a spongy layer of cytoplasmic tubules which extend from the epithelial cells. Sensory endings of the olfactory receptors are entirely within this spongy layer. Aquatic pulmonates may have a similar spongy layer in their olfactory epithelia, but the cilia of sensory endings, as well as motile cilia of epithelial cells, extend well beyond the spongy layer.     

Microvasculature of the buffalo (Bubalus bubalis) choroid plexuses: structural, histochemical, and immunocytochemical study

The choroid plexuses (CPs) in mammals produce the cerebrospinal fluid (CSF). In the literature, the morphology of CPs and the process that regulates the production of CSF are virtually nonexistent for domestic ruminants. Thus this study has two aims: 1. to investigate the morpho-structure of the buffalo CP microvasculature utilizing light microscopy (LM), scanning electron microscopy (SEM) and transmission electron microscopy (TEM) techniques, and 2. to investigate the relationship between the blood vessels and both the elongated cells and the cells with multiple protrusions located in the CPs. SEM and TEM analyses of the CPs from buffalo brain showed morphological and structural features similar those reported in other mammalian species. Moreover the blood microvasculature is the major component responsible for the formation of the CSF, secreted by the encephalic CPs. In addition the chemical composition of this fluid depends on several morpho-functional characteristics of the vascularization of the CPs. These characteristics are as follows: two shapes of the vascular organization: lamina-like and ovoid-like elongated cells of the CPs, which connect the ventricular cavities to the blood capillaries; and the CP capillaries have diverse forms. In the present study the employment of NADPHd and NOS I was taken as indirect evidence for the presence of NO for investigation their specific role in CPs. Then NOS I immunoreactivity is found in the walls of CP blood vessels demonstrating indirectly the presence of NO with a vaso-dilatatory and autoregulation function of vascular tone by cholinergic nerve stimulation of blood vessel smooth muscle.     

Nitric oxide alleviates cadmium-impeded growth by limiting ROS accumulation in pea seedlings

Cadmium (Cd) causes oxidative stress, which leads to the oxidation of various biomolecules by the production of reactive oxygen species (ROS) to facilitate programmed cell death (PCD). The antioxidant defense system fails to detoxify ROS when it is produced in excess. Nitric oxide (NO), a gaseous free radical and a phytohormone, regulates various physiological processes of plants. Therefore, this work was undertaken to study the effects of the application of exogenous sodium nitroprusside (SNP, a NO donor) on growth parameters, oxidative stress, accumulation of secondary metabolites, and activities of antioxidant enzymes under Cd stress. Mild (50 µM) and severe (200 µM) Cd stress were applied to hydroponically grown pea (Pisum sativum L.) plants with or without 50 µM SNP. Severe Cd stress had a substantial impact on the plants. The effectiveness of NO in reducing Cd-induced negative effects on plant height, fresh weight, dry weight, protein content, nitrite content, nitrate reductase (NR) activity, catalase activity, and peroxidase activity were investigated. Seedling development, protein content, nitrite content, nitrate reductase (NR) activity, antioxidant defense systems disruption, overproduction of reactive oxygen species, and oxidative damage were observed. The antioxidant defense system (catalase and peroxidase activities) was activated by NO, which resulted in lower lipid peroxidation and lower hydrogen peroxide (H₂O₂) levels in Cd-exposed plants. SNP treatment boosted endogenous NO levels and NR activity in Cd-stressed plants while also enhanced proline levels to preserve osmotic equilibrium. The presence of total phenols and flavonoids increased after SNP treatment, indicating that SNP enhanced stress recovery and boosted plant development in Cd-stressed plants.

     

Nitric oxide: relation to integrity, injury, and healing of the gastric mucosa

Nitric oxide (NO) plays a multifaceted role in mucosal integrity. The numerous functions of NO and the double-edged role played by NO in most of them provide a great complexity to the NO action. The three enzymatic sources of NO, neuronal NO-synthase (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS), have been characterised in the gastrointestinal tract. The protective properties of the NO derived from constitutive NO-synthases (eNOS and nNOS) have already been well established. Less clear is the role assigned to iNOS. The simplistic initial view of low levels of NO synthesised by constitutive NOS being protective while exaggerated NO levels after iNOS induction leading irremediably to cytotoxicity is being questioned by new evidence. As initially reported for constitutive NOS, iNOS activity may be associated to reduced leukocyte-endothelium interaction and platelet aggregation as well as protection of mucosal microcirculation. Moreover, iNOS activity may be important to resolve inflammation by increasing apoptosis in inflammatory cells. It is entirely possible that a low level of expression of iNOS will reflect a positive host-defense response to challenge, but that exaggerated or uncontrolled expression of iNOS itself becomes detrimental. There is no doubt about the protective role of NO in physiological conditions. However, when the mucosa is threatened, the role of NO becomes multiple and the final effect will probably depend on the nature of the insult, the environment involved, and the interaction with other mediators.     

Structure, heterologous expression, and adhesive properties of the P(0)-like myelin glycoprotein IP1 of trout CNS

The IP1 protein of trout CNS myelin as well as an IP1/P(0) chimeric protein were stably expressed in CHO cells. Successful targeting of the recombinant proteins to the membrane surface was verified by immunofluorescence staining. Full-length expression of IP1 could be confirmed by Western blot analysis of proteins extracted from stably transfected CHO-cells. The adhesive properties of IP1 were studied by an in vitro aggregation assay in which microscopic examination was combined with electronic particle counting. While IP1 conveyed only a weak increase in cell aggregation of transfected CHO cells, the IP1/P0 chimera was much more effective. In the presence of specific antibodies, cell aggregation was strongly reduced. The adhesive properties of P(0)-like proteins are discussed considering recent crystallographic data on the atomic structure of the extracellular domain of mammalian P(0).     

Involvement of the choroid plexus in central nervous system inflammation

During inflammatory conditions in the central nervous system (CNS), immune cells immigrate into the CNS and can be detected in the CNS parenchyma and in the cerebrospinal fluid (CSF). The most comprehensively investigated model for CNS inflammation is experimental autoimmune encephalomyelitis (EAE), which is considered the prototype model for the human disease multiple sclerosis (MS). In EAE autoagressive CD4(+), T cells gain access to the CNS and initiate the molecular and cellular events leading to edema, inflammation, and demyelination in the CNS. The endothelial blood-brain barrier (BBB) has been considered the obvious place of entry for the circulating immune cells into the CNS. A role of the choroid plexus in the pathogenesis of EAE or MS, i.e., as an alternative entry site for circulating lymphocytes directly into the CSF, has not been seriously considered before. However, during EAE, we observed massive ultrastructural changes within the choroid plexus, which are different from changes observed during hypoxia. Using immunohistochemistry and in situ hybridization, we observed expression of VCAM-1 and ICAM-1 in the choroid plexus and demonstrated their upregulation and also de novo expression of MAdCAM-1 during EAE. Ultrastructural studies revealed polar localization of ICAM-1, VCAM-1, and MAdCAM-1 on the apical surface of choroid plexus epithelial cells and their complete absence on the fenestrated endothelial cells within the choroid plexus parenchyme. Furthermore, ICAM-1, VCAM-1, and MAdCAM-1 expressed in choroid plexus epithelium mediated binding of lymphocytes via their known ligands. In vitro, choroid plexus epithelial cells can be induced to express ICAM-1, VCAM-1, MAdCAM-1, and, additionally, MHC class I and II molecules on their surface. Taken together, our observations imply a previously unappreciated function of the choroid plexus in the immunosurveillance of the CNS.     

Measurements of NOx, acyl peroxynitrates, and NOy with automatic interference corrections using a NO2 analyzer and gas phase titration

NO(2) analyzers are much more valuable if they can also measure NO since the two (NO+NO(2)=NO(x)) are often found together. NO can be quantitatively converted to NO(2) by reaction with ozone and subsequent thermal decomposition of the N(2)O(5) that may form from further oxidation. The conversion of NO, along with decomposition of N(2)O(5) and removal of the remaining unreacted ozone with a heated chamber, allows for quantitative determination of NO(x) using a NO(2) analyzer and the determination of decomposed acyl peroxynitrates. Ambient tests are performed to demonstrate these methods.     

Cholinergic-nitrergic transmitter mechanisms in the cerebral circulation

Cerebral blood vessels from several species are innervated by vasodilator nerves. Acetylcholine (ACh) released from parasympathetic cholinergic nerves was first suggested to be the transmitter for vasodilation. Results from pharmacological studies in isolated cerebral arterial ring preparations, however, have demonstrated that nitric oxide (NO) but not ACh mediates the major component of neurogenic vasodilation. More recently, ACh and NO have been shown to co-release from the same cholinergic-nitrergic nerves, and that ACh acts as a presynaptic transmitter in modulating NO release. In this communication, evidence for the neuronal origin of NO and possible role of ACh in modulating NO release in large cerebral arteries at the base of the brain will be discussed.     

NO message from muscle.

The synthesis of the free radical gas nitric oxide (NO) is catalyzed by the enzyme NO synthase (NOS). NOS converts arginine and molecular oxygen to NO and citrulline in a reaction that requires NADPH, FAD, FMN, and tetrahydrobiopterin as cofactors. Three types of NOS have been identified by molecular cloning. The activity of the constitutively expressed neuronal NOS (nNOS) and endothelial NOS (eNOS) is Ca(2+)/calmodulin-dependent, whereas that the inducible NOS (iNOS) is Ca(2+)-insensitive. The predominant NOS isoform in skeletal muscle is nNOS. It is present at the sarcolemma of both extra- and intrafusal muscle fibers. An accentuated accumulation of nNOS is found in the endplate area. This strict sarcolemmal localization of nNOS is due its association with the dystrophin-glycoprotein complex, which is mediated by the syntrophins. The activity of nNOS in skeletal muscle is regulated by developmental, myogenic, and neurogenic influences. NO exerts several distinct effects on various aspects of skeletal muscle function, such as excitation-contraction coupling, mitochondrial energy production, glucose metabolism, and autoregulation of blood flow. Inside the striated muscle fibers, NO interacts directly with several classes of proteins, such as soluble guanylate cyclase, ryanodine receptor, sarcoplasmic reticulum Ca(2+)-ATPase, glyceraldehyde-3-phosphate dehydrogenase, and mitochondrial respiratory chain complexes, as well as radical oxygen species. In addition, NO produced and released by contracting muscle fibers diffuses to nearby arterioles where it acts to inhibit reflex sympathetic vasoconstriction.     

The compact neutron spectrometer at ASDEX Upgrade

The first neutron spectrometer of ASDEX Upgrade (AUG) was installed in November 2008. It is a compact neutron spectrometer (CNS) based on a BC501A liquid scintillating detector, which can simultaneously measure 2.45-MeV and 14-MeV neutrons emitted from deuterium (D) plasmas and γ radiation. The scintillating detector is coupled to a digital pulse shape discrimination data acquisition (DPSD) system capable of count rates up to 10(6) s(-1). The DPSD system can operate in acquisition and processing mode. With the latter n-γ discrimination is performed off-line based on the two-gate method. The paper describes the tests of the CNS and its installation at AUG. The neutron emission from the D plasma measured during a discharge with high auxiliary heating power was used to validate the CNS performance. The study of the optimal settings for the DPSD data processing to maximize the n-γ discrimination capability of the CNS is reported. The CNS measured both 2.45-MeV and 14-MeV neutrons emitted in AUG D plasmas with a maximum count rate of 5.4 × 10(5) s(-1) (>10 times higher than similar spectrometers previously achieved) with an efficiency of 9.3 × 10(-10) events per AUG neutron.     

一氧化氮电化学传感器及其在生物医学中的应用

一氧化氮（NO）是一种含有可以调控不断生长的生物学过程的非共享电子对的气体自由基，它由一氧化氮合成酶家族的L-精氨酸所形成。NO在人体内分布广泛，是帮助机体抵抗心血管疾病与其他疾病的信号分子。缺乏NO可能导致糖尿病、心血管疾病与其他疾病，而补充NO可预防和逆转此类疾病。NO是非常小的分子，十分活泼，半衰期短，它可以进入细胞，并向周围的细胞发出交流信号。但是，要准确检测其在生物体中的含量很困难。目前直接用于一氧化氮检测的方法不多，电化学方法尤其是电化学传感器是应用广泛的一类方法，由于其操作简单，灵敏度高，选择性好，已成为现代生物医学中研究一氧化氮的重要工具。本文主要综述近年来NO电化学传感器的研制及其在生物医学中的应用。     

Hypoxia-induced differential apoptosis in the central nervous system of the sturgeon (Acipenser shrenckii)

Hypoxia is a frequent challenge to aquatic vertebrates as compared with that for their terrestrial counterparts. All vertebrates respond to hypoxia in a similar, but not identical manner, indicating that these responses appeared early in the evolution of vertebrates. The aim of this study is to find out the effects of hypoxia on apoptosis in the central nervous system (CNS) of sturgeon, an archaic fish. With the regional specialization of the CNS, we hypothesize that if cell death does occur, the response will vary between regions, i.e., some CNS areas will be more susceptible to hypoxia than the others would. Sturgeons (Acipenser shrenckii) were subjected to hypoxia by exposure to either air or hypoxic water. After 6- or 30-h recovery they were sacrificed and the following regions of the CNS: retina, olfactory lobe, optic tectum, pituitary, cerebellum, pons/medulla, and spinal cord were examined by the terminal transferase mediated dUTP nick end labeling technique and for the cleaved fragment of activated caspase-3 by Western blotting. In hypoxia-treated sturgeons, the retina, optic tectum, pituitary, and spinal cord were found to have significantly more apoptotic cells than did untreated sturgeons at both 6 and 30 h after the hypoxic insults, indicating prolonged damage. Apoptosis was confirmed by Western blotting of the cleaved fragment of activated caspase-3. Olfactory lobe, cerebellum, and pons/medulla had relatively few apoptotic cells. The CNS of sturgeon showed a differential pattern of apoptosis in response to hypoxia.     

Numerical study on no emission with flue gas dilution in air and fuel sides

Flue gas recirculation (FGR) is widely adopted to control NO emission in combustion systems. Recirculated flue gas decreases flame temperature and reaction rate, resulting in the decrease in thermal NO production. Recently, it has been demonstrated that the recirculated flue gas in fuel stream, that is, the fuel induced recirculation (FIR), could enhance much improved reduction in NO per unit mass of recirculated gas, as compared to conventional FGR in air. In the present study, the effect of dilution methods in air and fuel sides on NO reduction has been investigated numerically by using N3 and CO2 as diluent gases to simulate flue gases. Counterflow diffusion flames were studied in conjunction with the laminar flamelet model of turbulent flames. Results showed that CO2 dilution was more effective in NO reduction because of large temperature drop due to the larger specific heat of CO2 compared to N₂. Fuel dilution was more effective in reducing NO emission than air dilution when the same recirculation ratio of dilution gas was used by the increase in the nozzle exit velocity, thereby the stretch rate, with dilution gas added to fuel side.     

Gene expression in the supraoptic nucleus.

The magnocellular neurosecretory cells (MNCs) in the supraoptic nucleus (SON) express multiple kinds of genes, including not only the classical hormones arginine vasopressin (AVP) and oxytocin (OXT), but also other physiologically active substances including neuropeptides, their receptors, and nitric oxide (NO) synthase, the rate-limiting enzyme in the synthesis of NO under physiological condition. For example, osmotic stimuli such as dehydration and chronic salt loading cause a wide range of changes of the expression levels of the genes and marked induction of the expression of the genes in the SON. The expression of the NO synthase gene in the SON under physiological conditions is reviewed.     

Improvement and experimental validation of a multi-zone model for combustion and NO emissions in CNG fueled spark ignition engine

This article reports the experimental and theoretical results for a spark ignition engine working with compressed natural gas as a fuel. The theoretical part of this work uses a zero-dimensional, multi-zone combustion model in order to predict nitric oxide (NO) emission in a spark ignition (SI) engine. The basic concept of the model is the division of the burned gas into several distinct zones for taking into account the temperature stratification of the burned mixture during combustion. This is especially important for accurate NO emissions predictions, since NO formation is strongly temperature dependent. During combustion, 12 products are obtained by chemical equilibrium via Gibbs energy minimization method and nitric oxide formation is calculated from chemical kinetic by the extended Zeldovich mechanism. The burning rate required as input to the model is expressed as a Wiebe function, fitted to experimentally derived burn rates. The model is validated against experimental data from a four-cylinder, four-stroke, SI gas engine (EF7) running with CNG fuel. The calculated values for pressure and nitric oxide emissions show good agreement with the experimental data. The superiority of the multizone model over its two-zone counterpart is demonstrated in view of its more realistic in-cylinder NO emissions predictions when compared to the available experimental data.