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The paracellular pathway in the lepidopteran larval midgut: modulation by intracellular mediators
No full textThe features of the paracellular pathway, an important route for the transfer of ions and mols. in epithelia, are in insects still poorly investigated and it has not yet been elucidated how the septate junction (SJ) acts as a transepithelial barrier. In this study, some properties of the paracellular pathway of Bombyx mori larval midgut, isolated in Ussing chambers, were detd. and the modulation of SJ permeability by intracellular events disclosed. Diffusion potentials evoked by transepithelial gradients of different salts indicated that the junction bore weak neg. charges and that the paracellular pathway was selective with respect to ion charge and size. In std. conditions, the transepithelial resistance was 28.2 +- 2.1 W cm2, a value indicating that the midgut is a low resistance epithelium. The modulation of midgut SJ by typical enhancers of mammalian tight junction permeability known to act on the cytoskeleton was studied by measuring the shunt resistance and the lumen-to-hemolymph flux of sucrose. An increase of the intracellular level of cAMP and Ca2+ caused a significant decrease of the shunt resistance and an increase of SJ permeability. The attenuation of Ca2+ effect in the presence of the Ca channel blocker nifedipine indicated that the influx of external Ca2+ into the cytoplasm was important for the opening of the SJ, as well as the release of Ca2+ from the intracellular stores. [on SciFinder (R)
Multiple transport pathways for dibasic amino acids in the larval midgut of the silkworm Bombyx mori
No full textThe transport pathways for dibasic amino acids were investigated in brush border membrane vesicles (BBMV) from the anterior-middle (AM) and posterior (P) regions of Bombyx mori midgut. In the absence of K+, a low-affinity saturable transport of arginine in both AM- and P-BBMV (Km 1.01 mM, Vmax 4.07 nmol/7s/mg protein and Km 1.38 1mM, Vmax 2.26 nmol/7s/mg protein, respectively) was detected. Arginine influx was dependent on the membrane electrical potential (Δψ) and increased raising the alkalinity of the external medium from pH 7.2 to 10.6. Competition experiments indicated the following order of substrate affinity: arginine, homoarginine, NG-monomethylarginine, NG-nitroarginine>lysine≫omithine>cysteine>methionine. Leucine, valine and BCH (2-amino-2-norbomanecarboxylic acid) did not inhibit arginine influx. In the presence of external K+, the influx of arginine as a function of arginine concentration fitted to a complex saturation kinetics compatible with both a low-affinity and a high-affinity component. The latter (Km 0.035 mM, Vmax 2.54 nmol/7s/mg protein) was fully characterized. The influx rate had an optimum ar pH 8.8, was strongly affected by Δψ and was homogeneous along the midgut. The substrate affinity rank was: homoarginine>arginine, NG-monomethylarginine≫cysteine, lysine≫NG-nitroarginine>omithine>methionine. Leucine and amino acids with a hydrophobic side chain were not accepted. This system is also operative in the absence of potassium, with the same order of specificity bur a very low activity. Lysine influx is mediated by two more transport systems, the leucine uniport and the K+/leucine symport specific for amino acids with a hydrophobic side chain that recognizes lysine ar extravesicular pH values (pHout) exceeding 9. Both the uniport and the symport differ from the cationic transport systems so far identified in mammals because they are unaffected by N-ethylmaleimide, have no significant affinity for neutral amino acids in the presence of the cation and show a striking difference in their optimum pH
Modulation of leucine absorption in the larval midgut of Bombyx mori (Lepidoptera, Bombycidae)
No full textIn the larval midgut of Bombyx mori a K+-dependent transporter for leucine and amino acids with a hydrophobic side chain is responsible for the absorption of most essential amino acids. We investigated if a modulation of its activity occurred as a result of starvation or after hormonal treatments. We measured amino acid uptake in brush border membrane vesicles (BBMV) purified from the anterior-middle (AM) and posterior (P) regions of the midgut in fifth instar larvae. Silkworms were either starved or topically treated with low dosages of fenoxycarb, a molecule often used as a juvenile hormone mimic. The maximal uptake value of K+-driven leucine transport was increased in BBMV of AM- and P-midgut regions of starved larvae. The initial uptake rates of serine and glutamine, two amino acids transported by the same cotransporter as leucine, were also increased. Leucine kinetics proved that Vmax was the kinetic parameter modified by starvation in both midgut regions. Topical applications of fenoxycarb at a dose of 2.5 fg/larva immediately after the fourth ecdysis, induced an increase of leucine initial uptake rates and of intravesicular accumulation of leucine in both AM- and P-BBMV. Kinetic analysis of leucine uptake indicated again that Vmax was increased in BBMV from both midgut regions in treated larvae
Cell penetration and electrophysiological effects of an insect parvovirus in the midgut of Spodoptera frugiperda
No full textDensoviruses (DNV) are insect parvoviruses sharing with that group a non enveloped 20-25 nm icosaedric capsid and a single stranded DNA genome. Since they are lethal for several insects at larval stages, including agronomical pests and insects vector-borne diseases, the question of their use as biopesticides is revisited. As a model, we studied the interaction of Junonia coenia Densovirus (JcDNV) and one permissive host, Spodoptera frugiperda (Lepidoptera, Noctuidae). The larvae get infected by the oral route, ingesting viral particles contaminating food. The success of the infection first depends on the penetration of the virus through the midgut epithelium. In order to study this early step, we performed a cellular and physiological analysis of the entry of a Cy3-labelled JcDNV through S. frugiperda midgut in vitro.
Primary cultures of midgut cells were developed to detect the sites of penetration and the intracellular pathway followed by Cy3-JcDNV. The virus was unable to infect stem cells and differentiated goblet cells, while in incubated columnar cells, whether at an early phase of differentiation or fully differentiated, the virus was visible after 10 minutes in the basolateral membrane and in microvilli, and after 30 minutes within the cytoplasm. Virus particles were apparent as spots, suggesting that they could be distributed in intracellular compartments.
S. frugiperda midguts were then isolated from fifth instar larvae, mounted between Ussing chambers and incubated with JcDNV added to the luminal side of the epithelium. After 10 minutes of exposition, the virus induced a significant decrease of the paracellular electrical resistance, an indication that ions moved more rapidly through the aqueous channels formed by the intercellular junctions. The confocal image of whole-mount midguts, isolated and incubated for 10 minutes with luminal Cy3-JcDNV, clearly showed the presence of the virus in the enterocytes’ cytoplasm
Role of specific activators of intestinal amino acid transport in Bombyx mori larval growth and nutrition
No full textNutrient absorption and its modulation are critical for animal growth. In this paper, we demonstrate that leucine methyl ester (Leu-OMe) can greatly increase the activity of the transport system responsible for the absorption of most essential amino acids in the larval midgut of the silkworm Bombyx mori. We investigated leucine uptake activation by Leu-OMe in brush border membrane vesicles and in the apical membrane of epithelial cells in the midgut incubated in vitro. Moreover, the addition of this strong activator of amino acid absorption to diet significantly affected larval growth. Silkworms fed on artificial diet supplemented with Leu-OMe reached maximum body weight 12-18 h before control larvae, and produced cocoon shells up to 20% heavier than those of controls. The activation of amino acid absorption plays an essential role in larval development so that larval growth and cocoon production similar to controls reared on an artificial diet with 25% of dry mulberry leaf powder were observed in silkworms fed on an artificial diet with only 5% of mulberry powder
Uso di sonde fluorescenti per lo studio dell'attività e della regolazione del sistema di trasporto K+/aminoacidi neutri in Bombyx mori
No full textIl sistema di trasporto K+-dipendente per gli aminoacidi neutri, ampiamente studiato nelle larve di Bombyx mori L., è perfettamente adattato alle peculiari condizioni presenti nell’intestino medio larvale. Sebbene sia in grado di operare in assenza di K+, è notevolmente attivato dal gradiente di K+, dalla presenza di un potenziale elettrico transmembrana e da un pH esterno alcalino. Questo sistema di trasporto, che lega preferenzialmente leucina ma è responsabile anche dell’assorbimento della maggior parte degli aminoacidi essenziali, è sotto il controllo di diversi fattori. In particolare, abbiamo identificato una famiglia di molecole, di cui la più attiva è la leucina metilestere (LeuMe), in grado di aumentarne l’attività in vitro e di attivare per ingestione l’assorbimento degli aminoacidi in vivo, a conferma che questo processo influenza lo sviluppo dell’intero organismo. La conoscenza delle proprietà funzionali e dei processi regolativi dei sistemi di trasporto intestinale degli aminoacidi in B. mori è utile sia per comprenderne le esigenze nutrizionali durante lo sviluppo larvale, specialmente durante l’ultima età, caratterizzata dalla massiccia sintesi delle proteine della seta, che per la formulazione di adeguate diete artificiali. L’utilizzo di traccianti radioattivi ha rappresentato finora un efficace mezzo per lo studio dei meccanismi di trasporto transmembrana, ma questa tecnica comporta l’esposizione del ricercatore a rischio radiologico e la produzione di rifiuti la cui gestione è complessa e costosa. Poiché l’uptake K+-dipendente della leucina è associato al trasporto della carica netta positiva dello ione, abbiamo valutato se l’impiego del DisC3(5), una sonda fluorescente sensibile alle variazioni del potenziale elettrico di membrana, fosse una valida alternativa ai traccianti radioattivi per la misura del trasporto di aminoacidi e della sua regolazione, in vescicole di membrana di orletto a spazzola purificate dall’intestino medio larvale di B. mori. Dopo aver calibrato il segnale fluorescente, l’utilizzo di questa tecnica ha consentito di misurare l’uptake di diverse concentrazioni di leucina e di rilevare l’incremento del trasporto dovuto all’effetto della LeuMe. Questa tecnica ha permesso di evidenziare, per al prima volta, che l’attivatore è in grado di aumentare il trasporto anche della forma binaria K+-trasportatore, cioè in assenza di leucina. In conclusione la tecnica fluorimetrica, seppur meno sensibile dei traccianti radioattivi, risulta idonea per lo studio funzionale dei trasporti di membrana che comportano il movimento di cariche nette
Effetto del proctolin sull'attività intestinale delle larve di lepidottero
No full textAl fine di identificare nuovi insetticidi ecocompatibili che possano essere impiegati in alternativa ai pesticidi chimici tradizionali, si stanno studiando sia gli effetti indotti da prodotti genici tossici isolati da virus, microorganismi, funghi, piante e artropodi, sia le alterazioni fisiologiche che si presentano in seguito a somministrazione di peptidi endogeni che agiscono come fattori di regolazione. Tra questi, il pentapeptide proctolin, neurotrasmettitore/modulatore ad attività miotropica, è uno dei più studiati. La ricerca che abbiamo condotto mira a chiarire se il proctolin possa essere un buon candidato per il controllo dei Lepidotteri. Abbiamo quindi verificato se l’intestino larvale presenti recettori per il peptide a livello della muscolatura viscerale, questione ancora dibattuta in letteratura. A tale scopo, usando Bombyx mori come specie modello, abbiamo registrato in vitro le contrazioni spontanee dell’intestino posteriore in assenza o in presenza di proctolin. Si è osservato che il peptide è in grado di incrementare la frequenza delle contrazioni intestinali, anche se a concentrazioni molto superiori a quelle ormonali. Il proctolin sembra essere quindi in grado di legarsi a recettori presenti sulla muscolatura intestinale delle larve di lepidottero, che presentano però un’affinità per il peptide estremamente bassa.
Abbiamo poi determinato l’effetto della somministrazione orale di proctolin sulla fitness larvale, valutando le variazioni causate dal peptide sui parametri nutrizionali standard. Alla concentrazione di 0.18 mg/g larva, il proctolin induce una riduzione significativa dell’efficienza di conversione in massa corporea del cibo ingerito (ECI) e digerito (ECD), con una conseguente diminuzione del tasso di crescita (GR). La concentrazione di peptide attiva nel ridurre la crescita larvale di B. mori è però estremamente critica. Infatti, aumentando di circa dieci volte la dose di proctolin somministrata alle larve, vengono attivati meccanismi compensativi in grado di annullare completamente l’effetto finale sulla GR. Tali meccanismi sono correlati ad una stimolazione del comportamento alimentare della larva, che aumenta la quantità di cibo ingerito.
Anche nelle larve di Spodoptera littoralis trattate con la bassa concentrazione di proctolin si osserva, come in B. mori, una riduzione del tasso di crescita
What is the role of nanotechnology in diagnosis and treatment of metastatic breast cancer? Promising scenarios for the near future
Full text linkMetastatic breast cancer represents a diagnostic and therapeutic challenge due to tumor heterogeneity and to various physiological barriers that hinder drug delivery to the metastatic sites. To overcome these limitations, nanoformulated drugs have been developed and tested in preclinical studies, and few of them have been successfully translated into clinical practice. In particular, liposomal anthracyclines and nanoformulated albumin-bound paclitaxel have revealed an improved therapeutic index when compared to conventional chemotherapy, with significant reduction of drugs toxicity. Several strategies for nanoparticles engineering have more recently been explored to increase selectivity for tumor cells and to reach poorly accessible metastatic districts. Targeted nanoparticles, directed toward tumor markers and tissue-specific metastases, may provide effective devices in case of lowvascularized and small-sized metastases, thus paving the way for a real change in the natural history of metastatic disease. A number of targets have been identified and exploited for surface functionalization of different types of nanoparticles, which are currently undergoing preclinical studies. The aim of this review is to provide an overview of current nanotechnology applied to metastatic breast cancer diagnosis and treatment. Promising results encourage an upcoming translation of this research into clinical practice for an effective management of the disease in the near future
Nanoformulated Antiretrovirals for Penetration of the Central Nervous System: State of the Art
Full text linkThe central nervous system is a very challenging HIV-1 sanctuary. But, despite complete suppression of plasmatic viral replication with current antiretroviral therapy, signs of HIV-1 replication can still be found in the cerebrospinal fluid in some patients. The main limitation to achieving HIV-1 eradication from the brain is related to the suboptimal concentrations of antiretrovirals within this site, due to their low permeation across the blood–brain barrier. In recent years, a number of reliable nanotechnological strategies have been developed with the aim of enhancing antiretroviral drug penetration across the blood–brain barrier. The aim of this review is to provide an overview of the different nanoformulated antiretrovirals, used in both clinical and preclinical studies, that are designed to improve their delivery into the brain by active or passive permeation mechanisms through the barrier. Different nanotechnological approaches have proven successful for optimizing antiretrovirals delivery to the central nervous system, with a likely benefit for HIV-associated neurocognitive disorders and a more debated contribution to the complete eradication of the HIV-1 infection
Transport of L-proline in different cell types of Homarus americanus (Crustacea Decapoda) hepatopancreas
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