New insights into N-utilization efficiency in tomato (Solanum lycopersicum L.) under N limiting condition.

Understanding Nitrogen Use Efficiency (NUE) physiological and molecular mechanisms in high N demanding crops has become decisive for improving NUE in sustainable cropping systems. How the Nitrogen Utilization Efficiency (NUtE) component contributes to the NUE enhancement under nitrate limiting conditions in tomato remains to be elucidated. This study deals with the changes in several important nitrate metabolism related gene expressions (nitrate assimilation, transport, remobilization and storage/sequestration) engendered by short and long-term limiting nitrate exposure in two selected NUE-contrasting genotypes, Regina Ostuni (RO) and UC82, efficient and inefficient, respectively. At short-term, nitrate limiting supply triggered higher SlCLCa and SlNRT1.7 expressions in RO root and shoot, respectively, suggesting a higher nitrate storage and remobilization compared to UC82, explaining how RO withstood the nitrate deficiency better than UC82. At long-term, nitrate reductase (SlNR) and nitrite reductase (SlNIR) expression were not significantly different between nitrate treatments in RO, while significantly down-regulated under nitrate limiting treatment in UC82. In addition, SlCLCa and SlNRT1.8 transcript levels were significantly lower in RO, while those of SlNRT1.5 and SlNR appeared significantly higher. This suggested that the efficient genotype stored less nitrate compared to UC82, which was allocated and assimilated to the shoot. More interestingly, the expression of SlNRT2.7 was significantly higher in RO shoot compared to UC82 and strongly correlated to RO higher growth as well as to NUE and NUtE component. Our findings underlined the differential regulation of N-metabolism genes that may confer to NUtE component a pivotal role in NUE enhancement in tomato.

Carbonic anhydrase integrated into a multimarker approach for the detection of the stress status induced by pollution exposure in Mytilus galloprovincialis: A field case study.

The work was addressed to study the sensitivity of the enzyme carbonic anhydrase (CA) to chemical pollution in the hepatopancreas of the bioindicator organism Mytilus galloprovincialis in the context of a multimarker approach in view of ecotoxicological biomonitoring and assessment application. The study was carried out by means of a transplanting experiment in the field, using caged organisms from an initial population exposed in the field in two areas of interest: Augusta-Melilli-Priolo, an heavy polluted industrial site (eastern Sicily, Italy), and Brucoli (eastern Sicily, Italy) an area not affected by any contamination and selected as a reference site. Mussels in Augusta presented a significant increase in the digestive gland CA activity and gene expression compared to the animals caged in the control site of Brucoli. The CA response in animals from the polluted site was paralleled by proliferation/increase in the size of lysosomes, as assessed by Lysosensor green charged cells, induction of metallothionein, up-regulation of hif-α (hypoxia-inducible factor), metabolic changes associated with protein metabolism, and changes in the condition factor. Biological responses data were integrated with information about sediment chemical analysis and metal residue concentration in animal soft tissues. In conclusion, obtained results highlighted the induction of CAs in the hepatopancreas of Mytilus galloprovincialis following to pollution exposure, and demonstrated its suitability to be integrated into a multimarker approach for the detection and characterization of the stress status induced by pollution exposure in this bioindicator organism.

Coumarin enhances nitrate uptake in maize roots through modulation of plasma membrane H+ -ATPase activity.

Coumarin is one of the simplest plant secondary metabolites, widely distributed in the plant kingdom, affecting root form and function, including anatomy, morphology and nutrient uptake. Although, some plant responses to coumarin have been described, comprehensive knowledge of the physiological and molecular mechanisms is lacking. Maize seedlings exposed to different coumarin concentrations, alone or in combination with 200 µm nitrate (NO3- ), were analysed, through a physiological and molecular approach, to elucidate action of coumarin on net NO3- uptake rate (NNUR). In detail, the time course of NNUR, plasma membrane (PM) H+ -ATPase activity, proton pumping and related gene expression (ZmNPF6.3, ZmNRT2.1, ZmNAR2.1, ZmHA3 and ZmHA4) were evaluated. Coumarin alone did not affect nitrate uptake, PM H+ -ATPase activity or transcript levels of ZmNRT2.1 and ZmHA3. In contrast, coumarin alone increased ZmNPF6.3, ZmNAR2.1 and ZmHA4 expression in response to abiotic stress. When coumarin and NO3- were concurrently added to the nutrient solution, a significant increase in the NNUR, PM H+ -ATPase activity, together with ZmNAR2.1:ZmNRT2.1 and ZmHA4 expression was observed, suggesting that coumarin affected the inducible component of the high affinity transport system (iHATS), and this effect appeared to be mediated by nitrate. Moreover, results with vanadate, an inhibitor of the PM H+ -ATPase, suggested that this enzyme could be the main target of coumarin. Surprisingly, coumarin did not affect PM H+ -ATPase activity by direct contact with plasma membrane vesicles isolated from maize roots, indicating its possible elicitor role in gene transcription.

Metallothioneins and heat shock proteins 70 in Armadillidium vulgare (Isopoda, Oniscidea) exposed to cadmium and lead.

The heavy metals bioaccumulation capability in Armadillidium vulgare feeded with chestnut leaves contaminated with various sublethal concentrations of Cd and Pb, was evaluated under laboratory conditions. The metal concentration found in the hepatopancreas of treated animals, as measured by Inductively Coupled Plasma Mass Spectrometry (ICP-MS), affected the expression and localization of MT and HSP70 as shown by immunohistochemical and western blotting analysis. The Cd content of the animals treated with the various concentrations of the metal has been always higher than that of chestnut leaves contaminated. The accumulation of Pb was, instead, always modest compared to the content of the chestnut leaves. The immunohistochemical investigation in hepatopancreas tissue of animals treated with increasing concentrations of Cd and Pb, by using the anti-MT and anti-HSP70 antibodies, has provided a response clearly positive even if differentiated in relation to the metal and concentration tested. In particular, a positive response to anti-MT antibody was detected in B and S cells nuclei and S cells cytoplasm; the localization of HSP70 was particularly intense at the cell surface. Western blotting analysis showed significant up-regulation of the expression (about 2.6 fold) of HSP70 proteins in the hepatopancreas of animals exposed to highest Pb concentrations respect to control. Moreover, samples exposed to higher Cd and Pb concentrations showed a higher expression of MT (3.2 fold and 4 fold respectively) compared to control. In summary, our data beyond to clearly demonstrate for the first time the expression of MT in terrestrial isopods, suggest that A. vulgare would be a suitable organism for assessing Cd and Pb exposure in environments threatened by metal pollution as suggested by the modulation of the biomarkers MT and HSP70.

Expression of metallothionein mRNAs by in situ hybridization in the gills of Mytilus galloprovincialis, from natural polluted environments.

Metallothioneins (MTs), metal-inducible proteins, are crucial proteins for the regulation of essential metals, and are transcriptionally induced in all organisms by certain heavy metals, oxidative stress and inflammation. The gills represent an organ of uptake and loss of metals in which different mechanisms are present controlling the functions directly involved in the maintenance of homeostasis. In this study, the morphological and histomorphological aspects of branchial epithelium in Mytilus galloprovincialis from polluted environment (Faro swamp, Messina, Italy) have been investigated. The reverse transcriptase-polymerase chain reaction (PCR) has been used to isolate complementary DNA of both MT isoforms present from RNA extracted from mussel gills. The respective mRNAs on histological sections have been visualized by in situ hybridization. These methods showed that MT-10 mRNA is expressed at the basal level. In contrast, the MT-20 expression level was very low under basal conditions, while its mRNA increased dramatically in individuals collected in Faro. The presence of acid mucocytes and MTs in the gills may be considered a further defensive mechanism also related to the significantly higher concentration of Cd, Pb and Cr found in gills of M. galloprovincialis from Faro than specimens from the reference site (Goro). The results obtained show that, in stressed mussels, the defensive processes increase to maintain the normal functions of the organs more exposed to the action of polluted substances.

Apoptosis, cell proliferation and serotonin immunoreactivity in gut of Liza aurata from natural heavy metal polluted environments: preliminary observations.

In the present paper, the effect of natural environment non-lethal heavy metal concentration on cell renewal of Liza aurata intestinal epithelium, was studied by the TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick end labelling) method and anti-PCNA (proliferating cell nuclear antigen) immunohistochemistry, in order to detect, respectively, apoptosis and cell proliferation. In addition, the presence and distribution of the cell renewal regulator, serotonin, was immunohistochemically investigated. In order to reduce variability, only immature specimens were considered. The results indicated that in the control specimens from non-polluted areas, the PCNA immunoreactive nuclei of the proximal intestinal epithelium were only located at the bottom of the intestinal folds, together with a few TUNEL-positive nuclei, and goblet mucous differentiated cells. In the specimens from polluted areas, the number of PCNA immunoreactive cells was greatly enhanced, and they extended along the mid portion of the intestinal folds; the number of TUNEL-positive nuclei was enhanced as well, but they were almost exclusively detected in the third apical portion of the intestinal folds. Serotonin immunoreactive nerve elements were more frequently detected in the intestinal wall of L. aurata specimens from polluted areas, and besides that, some serotonin immunoreactive endocrine cells were also present. Variations in distribution and frequency of TUNEL-positive nuclei, PCNA immunoreactive nuclei, and serotonin immunoreactivity put in evidence an alteration of cell renewal with an enhancement of cell proliferation, probably leading to morphological intestinal fold changes.

Immunohistochemical investigation of the nitrergic system in the taste organ of the frog, Rana esculenta.

We have studied by immunocytochemistry, the taste discs of the frog, Rana esculenta, with the aim of providing morphological and neurochemical data on the nitrergic system and of assessing the eventual presence of intrinsic neurons associated with the gustatory organs. In taste discs, antibodies against neuronal nitric oxide synthase (nNOS) revealed a positive immunoreaction in the taste receptor cell bodies and processes. The basal cells were also stained. All the fungiform papillae contained intragemmal nerve fibers showing nNOS immunoreactivity; these fiber were mainly located in the basal plexus. Immunoreactive nerve fibers were also visible at the periphery of the papilla-contacting ciliate cells, which form a ring around the taste disc. In conclusion, the findings obtained in this study suggest that the occurrence of nNOS-immunoreactivity in basal cells, taste cells and nerves might reflect a role for nitric oxide in taste mechanisms of Amphibia. The results may also sustain the physiological implication of NO as a molecule involved in the local target function of maintaining the taste bud mucosal integrity and in regulating the blood flow to the epithelium.

Lectin histochemistry of epidermal glandular cells in the earthworm Lumbricus terrestris (Annelida Oligochaeta).

Carbohydrate residues were localized in the glandular cells of the epidermis of Lumbricus terrestris by lectin histochemistry. The following biotinylated lectins were used: ConA, PNA, WGA, UEA-I. Each lectin has a specific binding pattern in the epidermal glandular cells. The ConA binding is evident in the orthochromatic mucous cells; PNA in the metachromatic mucous cells; WGA in the neuroendocrine-like cells; UEA-I in the cuticle. The epidermal glandular cells possess specific sites for the different lectins in relation to their functional characteristics. Therefore, these sugar residues indicate different behaviours of the cells in epidermal functions related to ion transport, receptor-secretory processes and defence.

Immunohistochemical localization of calcium-binding proteins (CaBPs) in the epidermis of the earthworm Lumbricus terrestris (Annelida, Oligochaeta).

The present immunohistochemical study provides the first evidence of the presence of calcium-binding proteins (CaBPs) in the epidermis of the earthworm Lumbricus terrestris (Annelida, Oligochaeta) a lower invertebrate. The entire epidermis was labelled for calmodulin which is in agreement with its ubiquitous occurrence. Immunopositivity for calbindin D28K was limited to mucous cells, while that for S-100 protein was present only in neuroendocrine-like small granular cells. Finally, labelling for parvalbumin was specifically present in the subcutaneous nerve plexus. S-100 protein is considered to be a marker of neuroendocrine cells, at least in lower invertebrates such as Annelida. Although calbindin D28K is considered to be a marker of these cells in vertebrates, the same function cannot be attributed in Lumbricus terrestris. However, we can conclude that S-100 protein, as a regulatory protein, is phylogenetically older than calbindin D28K. We assume that the latter has an autoregulatory function in secretory processes. In agreement with previous data, we suggest that small granular cells exert a paracrine action in osmoregulatory and secretory processes.

Neuronal nitric oxide synthase (nNOS) expression in the epithelial neuroendocrine cell system and nerve fibers in the gill of the catfish, Heteropneustes fossilis.

We studied immunohistochemically the localization of neuronal nitric oxide synthase (nNOS) in gills of an Indian catfish species, Heteropneustes fossilis. It is shown that most of the epithelial neuroendocrine cells that are present in gill filaments and lamellae stained positively. Co-localization of nNOS and endothelin was also shown in neuroendocrine cells. A dense plexus of nNOS-containing nerve fibers was present beneath the gill epithelium, associated with efferent filament arteries and the basal side of neuroendocrine cells. nNOS immunopositive neurons were not found in gill areas. nNOS immunopositive neuroendocrine cells appeared to differ from neuroepithelial cells in gills of various teleost species, which are considered as oxygen-sensitive receptors and are present in the distal halves of gill filaments. Other types of neuroendocrine cells have been identified previously in other areas of gills using antibodies to serotonin and endothelin peptides. These cell types are likely to be involved in chemical regulation of the physiology of gill cells. In relation to the function of the other cell types, our data on nNOS localization suggest that NO is a wide-spread transmitter in the gill of the Indian catfish. It may play a role both in the local regulation of vascular tone and in inhibitory innervation of the gill.

Lectin binding patterns in amphibian skin epithelium.

Seven lectins (PNA, DBA, WGA, UEA-I, RCA, SBA, Con A) were used to localize glycoconjugates in the skin of 10 species of Amphibia, 7 anurans (Bufo marinus, Bufo bufo, Rana ridibunda, Rana pipiens, Hyla arborea, Pelobates syriacus and Xenopus laevis) and 3 urodeles (Salamandra salamandra, Triturus vulgaris and Ambystoma mexicanum). It was found that every lectin has a specific binding pattern in the skin of each species. No common pattern could be established, either among frogs or toads, nor for a particular lectin. Each lectin bound specifically and selectively to a particular epithelial component, which differed from one species to the other. A number of lectins showed selective binding to mitochondria-rich cells, but, again, a pattern in positivity could not be found. It is concluded that lectin histochemistry does correlate with cellular function. Our data can be applied in studies of epithelium and skin development, and of changes that occur during adaptation to the environment by amphibian species.

Immunohistochemical localization of bioactive substances in the epidermis of the earthworm, Lumbricus terrestris (Annelida, oligochaeta).

Serotonin (5HT), endothelin-big (ET-big) and neuron-specific enolase (NSE) are present in some epithelial paraneuronal cells of vertebrates. In the invertebrates, we show for the first time, by immunofluorescence and immunoperoxidase methods, the immunoreactions to antibodies raised to ET, 5-HT and NSE in the epidermis of the Lumbricus terrestris. It is assumed that the reactive cells are small granular cells that may have a paracrine action in the mechanisms of cell proliferation and secretory processes. The function of these bioactive substances in the epidermis of the earthworm awaits investigation. Phylogenetically it is very interesting that paraneuronal-like type of cells appear in such an ancient species of invertebrate which uses the epidermis for various functional activities.

Lectin binding pattern and band 3 localization in toad skin epithelium and the effect of salt acclimation.

Seven lectins were employed to localize glycoconjugates in the skin of a toad (Bufo viridis). Each of the lectins exhibited a particular, specific and selective binding pattern. Peanut lectin (PNA) and WGA bound to mitochondria-rich (MR) cells, but WGA bound also abundantly, in the dermis. Band 3-like protein, as indicated by the reaction with polyclonal anti band 3 antibody, was localized exclusively in MR cells. Ionic acclimation (200 mmol/L NaCl, or 50 mmol/L KCl) affected profoundly the binding pattern of the lectins. High NaCl acclimation resulted also in diminishing anti band 3 antibody binding, whereas in skins of KCl-acclimated toads the staining remained similar to the control. The binding of WGA but not PNA, corresponded with the same cells that stained with anti band 3 antibody. PNA in concentration of > 10 micrograms/mL reduced reversibly, both the resting and activated Cl- conductance by 25-30%. Based on differential binding of band 3, WGA and PNA, these observations provide conclusive verification of the presence of at least two populations of MR cells in the toad skin epithelium. It is suggested that the PNA positive MR cells may correspond to a beta-type MR cell. The information can be used to study molecular mechanisms that are involved in ionic acclimation.

Temporal versus superior approach phacoemulsification: short-term postoperative astigmatism.

PURPOSE: To compare short-term clinical, postoperative astigmatism, and uncorrected visual acuity results 2 months after no-stitch phacoemulsification using a temporal clear corneal approach and poly(methyl methacrylate) (PMMA) intraocular lens (IOL) implantation through a 5.2 mm incision with those after no-stitch superior corneoscleral phacoemulsification. SETTING: University Eye Clinic of Palermo, Italy. METHODS: This prospective clinical trial comprised 80 patients with senile cataract who were randomly assigned to have phacoemulsification using a temporal approach or a superior approach. Surgically induced astigmatism (SIA) was calculated using Naeser's polar value method for determining with-the-rule (WTR) or against-the-rule (ATR) change. RESULTS: There was a highly significant between-group difference in SIA at each examination (P < .001; t-test) because of positive (WTR) SIA in the temporal approach group and negative (ATR) SIA in the superior approach group. No other significant differences between groups were found (P < .05, t-test). More patients in the temporal group had an uncorrected visual acuity of 20/25 or better 2 weeks postoperatively, although the difference between groups was not statistically significant (P = .562; chi-square). CONCLUSION: Phacoemulsification using a no-stitch, temporal, 5.2 mm clear corneal incision produced results comparable to those using a corneoscleral superior approach. A longer study of a larger series should be done to confirm the findings.

Immunocytochemical detection of islet hormones in the digestive system of Protopterus annectens.

The presence, distribution, and interrelationships of the four typical pancreatic islet hormones were investigated in the digestive system of Protopterus annectens by single and double immunohistochemical methods. Insulin-, glucagon-, and somatostatin-immunoreactive (IR) elements were detected in both the pancreas and the gut. Pancreatic polypeptide (PP)-IR endocrine cells were always present in the gut, but were only present in the pancreas of a few specimens. Some of the latter cells also seemed to react with glucagon antiserum. In the pancreas the immunopositive cells were organized into islets of different sizes, and their organizations were studied by the double immunohistochemical techniques. In the few large islets insulin-IR cells were present in the central zone, glucagon- and PP-IR cells at the periphery, and somatostatin-IR cells intermingled with both the peripheral and the central endocrine cells. In the smaller islets, the number and the staining intensity of glucagon- and PP-IR endocrine cells varied markedly. In the gut, insulin-, somatostatin-, and PP-IR cells were of the open type; glucagon-containing cells were very few and had no luminal contact. They were differently distributed along the intestinal epithelium. Somatostatin-IR nerve fibers and somatostatin-IR neuron cell bodies were also observed in the intestinal wall. The organization of pancreatic endocrine cells in P. annectens is similar to that observed in the majority of teleosts even if a different topographical association can be found. Furthermore, islets of different sizes seem to display a different metabolic turnover, and the detection of pancreatic PP-immunoreactivity varied according to the specimens utilized. In the intestinal portion insulin-IR cells, in addition to PP-, glucagon- and somatostatin-IR cells are present: this suggests that intestinal insulin-like immunoreactivity may be more widespread than previously supposed.

Localization of immunoreactive endothelin in the neuroendocrine cells of fish gill.

Immunohistochemical tests have demonstrated for the first time the presence of endothelins in the neuroendocrine cells of fish gill. We have sought co-localization of endothelins with serotonin and neuropeptides which are regarded as neuroendocrine markers of pulmonary diffuse neuroendocrine systems in higher vertebrates. Regarding their endocrine and paracrine activities in mammals, endothelins are considered as peptide hormones and growth factors regulating respiratory function. The roles of endothelins in the gill await investigation based on the multifunctional organization of this organ.

Enkephalin immunoreactivity in the paraneurons of the tiger salamander (Ambystoma tigrinum) tongue.

Immunohistochemical tests have demonstrated the presence of leu-5-enkephalin and other bioactive compounds (serotonin and neuron-specific enolase) in the basal cells of lingual taste buds in Ambystoma tigrinum; there was also a weak reaction for met-5-enkephalin. Similar reactions were obtained from particular cells dispersed within the lingual epithelium, which are provisionally identified as Merkel cells.

Neuroendocrine cells in the gills of the bowfin Amia calva. An ultrastructural and immunocytochemical study.

Neuroendocrine (NE) cells were localized by electron microscopy and immunocytochemistry in the gill epithelium of bowfin Amia calva. The NE cells are dispersed in whole epithelium of the gill as solitary cells without intraepithelial innervation. All the observed NE cells do not reach the surface of the epithelium. The NE cells are characterized by a large nucleus with patches of condensed chromatin, numerous mitochondria, a well developed Golgi apparatus and a few dense core vesicles of various size scattered in the cytoplasm. Dense core vesicles range from 100 to 560 nm in diameter, while a clear space between the electron dense core ant the limiting membrane ranges from 20 to 240 nm. Immunocytochemical observations reveal the presence of general neuroendocrine markers such as neuro-specific enolase and bioactive substances: serotonin, leu-enkephalin and met-enkephalin. we demonstrated the presence of endothelin - for the first time in fish - and suggested a local paracrine role for the NE cells. Some ultrastructural aspects and the immunocytochemical characteristics of NE cells of bowfin gills are common with those encountered in such cells of other lower vertebrate species.

Distribution patterns of cytokeratins in epidermis and horny teeth of the adult sea lamprey, Petromyzon marinus.

An immunohistochemical characterization of cytokeratins in the skin tissues of the sea lamprey, Petromyzon Marinus was performed using a panel of monoclonal antibodies. Cytokeratins typical for simple epithelia have been detected in the epithelial cells, with a 8/18 pair expression. Granular cells and skein cells showed a labelling of cytokeratins 7, 8, 18 and 19, that is observed also in the non-keratinized layers of the horny teeth. Similar cytokeratins occur in the outermost cell layers of the epidermis; cytokeratin 19 shows a comparatively weaker reaction. These results suggest that the cytokeratin patterns in the above types of cells of adult epidermis are quite different from those in ammocoetes where the expression of cell specific cytokeratins may be correlated with specific programs of epidermal differentiation.

Cytokeratin type distribution in the skin and gill epithelia of the Indian freshwater catfish, Heteropneustes fossilis as detected by immunohistochemistry.

Based on the general cross-reactivity of the cytokeratins in vertebrates, we describe the immunoreactivity for some mammalian cytokeratins of both the epidermis and gill of H. fossilis. The following monoclonal antibodies, commercially supplied, were tested: K8.13, KL1, AE1 and AE3, which have a wide spectrum of specificity, and LDS-68, M 20, K8.60, KS-B17.2, K4.62, which are more narrowly specific. The reaction of the epithelial cells of the skin to K8.13 was negative in the basal layer, weakly positive in the layers above but strongly positive in some cells of the superficial layer. KL1 was negative in the basal layer, positive in the outer layers. AE1 was strongly positive in the basal layer, negative in the superficial cells. AE3 gave a general but weak reaction in the epithelial cells. K8.60 was negative for the epithelial cells, but reacted positively in the club cells. Club cells also reacted to K8.13 and AE1, and were strongly positive to KL1 and AE3. Goblet mucous cells were negative to all antibodies tested. In the gills, K8.13 labelled cells of both filament and lamellar epithelia. The reaction to AE3 was similar but less intense. KL1 was positive in the basal layer of the filament epithelium but not elsewhere, and K8.60 was negative throughout. AE1 and KS- B17.2 specifically labelled small cells scattered in the filament and lamellar epithelia, which are tentatively identified as neuroendocrine cells.