The relevant adsorption of cis-[Pt(NH3)2(P2O7)](2-) (phosphaplatin) on hydroxyapatite nanocrystals (nHAP) was observed and studied in water suspension. Phosphaplatin cytotoxicity, which is very low for HeLa, MCF-7 and HS-5 cell lines could be enhanced, reaching that of cisplatin, by interaction with solid nHAP. This effect stems from nHAP ability to catalyze the phosphaplatin hydrolysis, producing the same hydrolytic species responsible for cisplatin antitumor activity.

Carnosine is an endogenous dipeptide abundant in the central nervous system, where by acting as intracellular pH buffering molecule, Zn/Cu ion chelator, antioxidant and anti-crosslinking agent, it exerts a well-recognized multi-protective homeostatic function for neuronal and non-neuronal cells. Carnosine seems to counteract proteotoxicity and protein accumulation in neurodegenerative conditions, such as Alzheimer’s Disease (AD). However, its direct impact on the dynamics of AD-related fibril formation remains uninvestigated. We considered the effects of carnosine on the formation of fibrils/aggregates of the amyloidogenic peptide fragment Aβ1-42, a major hallmark of AD injury. Atomic force microscopy and thioflavin T assays showed inhibition of Aβ1-42 fibrillogenesis in vitro and differences in the aggregation state of Aβ1-42 small pre-fibrillar structures (monomers and small oligomers) in the presence of carnosine. in silico molecular docking supported the experimental data, calculating possible conformational carnosine/Aβ1-42 interactions. Overall, our results suggest an effective role of carnosine against Aβ1-42 aggregation.

Cisplatin, cis-[Pt(NH3)2Cl2], was the first inorganic compound applied in clinics to treat a broad range of malignancies. It is able to bind DNA on the N7 positions of adjacent G/A residues, resulting in the cross-link lesions believed to be responsible for observed antitumor activity. Sometimes N7-metalated purines seem to be characterized by a relevant antitumor activity. This has led us to hypothesize a parallel mechanism of action of platinum drugs, based on free platinated purines, formed after drug administration and incorporated into DNA by DNA polymerases. In order to evaluate this possibility, as a key step to develop new drugs, we performed experiments focused on platinated nucleobases, i.e. [Pt(dien)(N7-G)] and cis-[Pt(NH3)2(py)(N7-G)], dien = diethylenetriamine, py = pyridine, G = 5’-dGTP, cell and mitochondrial uptake and processing. For the first time cell uptake and mobility mechanisms, related to plasmatic cell and/or mitochondrial membrane crossing, has been studied. The results of the present study suggest that nucleotide carriers can be actively implicated in the specific uptake of free cytoplasmic platinum bonded nucleotides. Moreover the possible insertion of metalated nucleobases into nuclear and/or mitochondrial new synthesized DNA/RNA, operated by DNA/RNA polymerases, has been evaluated.

Large bone or osteochondral defects still need new approaches to ameliorate the regeneration process. The integration of magnetic nanoparticles into synthetic/natural scaffold formulations, could lead to obtain a suitable, responsive “on demand” tool able to guide the regeneration process. The aim of this work was the design and characterization of chitosan-based scaffolds containing dextran-grafted maghemite (DM) with modular mechano-structural and biomimetic properties implemented by the presence of a bioactive agent such the L-arginine amino acid. Both components can act as modulators of the scaffold features and, at the same time, the simultaneous presence of MNPs and L-Arg can be exploited to induce variations with respect to the cytocompatibility responses.

Autosomal recessive spastic ataxia of CharlevoixSaguenay (ARSACS) is a neurological disease with mutations in SACS, encoding sacsin, a multidomain protein of 4,579 amino acids. The large size of SACS and its translated protein has hindered biochemical analysis of ARSACS, and how mutant sacsins lead to disease remains largely unknown. Three repeated sequences, called sacsin repeating region (SRR) supradomains, have been recognized, which contribute to sacsin chaperone-like activity. We found that the three SRRs are much larger (1,100 residues) than previously described, and organized in discrete subrepeats. We named the large repeated regions Sacsin Internal RePeaTs (SIRPT1, SIRPT2, and SIRPT3) and the subrepeats sr1, sr2, sr3, and srX. Comparative analysis of vertebrate sacsins in combination with fine positional mapping of a set of human mutations revealed that sr1, sr2, sr3, and srX are functional. Notably, the position of the pathogenic mutations in sr1, sr2, sr3, and srX appeared to be related to the severity of the clinical phenotype, as assessed by defining a severity scoring system. Our results suggest that the relative position of mutations in subrepeats will variably influence sacsin dysfunction. The characterization of the specific role of each repeated region will help in developing a comprehensive and integrated pathophysiological model of function for sacsin.

Solute Carrier 15 (SLC15) family, alias H(+)-coupled oligopeptide cotransporter family, is a group of membrane transporters known for their role in the cellular uptake of di- and tripeptides (di/tripeptides) and peptide-like molecules. Of its members, SLC15A1 (PEPT1) chiefly mediates intestinal absorption of luminal di/tripeptides from dietary protein digestion, while SLC15A2 (PEPT2) mainly allows renal tubular reabsorption of di/tripeptides from ultrafiltration, SLC15A3 (PHT2) and SLC15A4 (PHT1) possibly interact with di/tripeptides and histidine in certain immune cells, and SLC15A5 has unknown function. Our understanding of this family in vertebrates has steadily increased, also due to the surge of genomic-to-functional information from 'non-conventional' animal models, livestock, poultry, and aquaculture fish species. Here, we review the literature on the SLC15 transporters in teleost fish with emphasis on SLC15A1 (PEPT1), one of the solute carriers better studied amongst teleost fish because of its relevance in animal nutrition. We report on the operativity of the transporter, the molecular diversity, and multiplicity of structural-functional solutions of the teleost fish orthologs with respect to higher vertebrates, its relevance at the intersection of the alimentary and osmoregulative functions of the gut, its response under various physiological states and dietary solicitations, and its possible involvement in examples of total body plasticity, such as growth and compensatory growth. By a comparative approach, we also review the few studies in teleost fish on SLC15A2 (PEPT2), SLC15A4 (PHT1), and SLC15A3 (PHT2). By representing the contribution of teleost fish to the knowledge of the physiology of di/tripeptide transport and transporters, we aim to fill the gap between higher and lower vertebrates.

The effects of dietary inclusions of size-fractionated peptides and free amino acids (FAAs) on Peptide Transporter 1 (PepT1) mRNA levels were assessed along the length of the intestine of juvenile Atlantic cod (Gadus morhua). Five groups of fish (10-15g) were fed for 46days on diets containing approximately 42% protein, provided either as fish meal (FM, control diet) or as a combination of FM with whole fish hydrolysate (FH), retenate after ultrafiltration of FH (UFR), nanofiltered retenate of FH (NFR), or a mix of FAAs, at a 30% level of FM substitution. PepT1 mRNA expression was assessed in pyloric caeca (S1) and the remainder of the intestine divided into four equally long segments (S2-S5). PepT1 transcripts were found in all segments, indicating that the whole intestine is involved in peptide absorption. Differences in the regional expression profile of PepT1 were found. Under control diet (FM diet) conditions, fish exhibited a reduced expression in S5 compared to S2. In fish fed FAA and UFR diets, PepT1 mRNA levels were higher in S2 and S3 compared to other regions. These data suggest that PepT1 may be variably recruited along the whole intestine, including the most distal part, in response to changes in the luminal protein source content. This adaptive response might be functional to keep a maximal efficiency of protein absorption at the intestinal level.

Collagen is one of the most used materials in scaffolding production; this is due to its peculiar characteristics that make the polymer highly biocompatible and efficient in regeneration induction and growth cone guidance. We aimed to investigate whether collagen could per se induce Schwann cell differentiation/proliferation and how it would do so. Results obtained in immortalized rat Schwann cells showed differential effects on several proliferation and differentiation markers depending on the type of collagen used to produce the scaffolds.

Defective dolichol-phosphate mannose synthase (DPMS) complex is a rare cause of congenital muscular dystrophy associated with hypoglycosylation of alpha-dystroglycan (α-DG) in skeletal muscle. We used the zebrafish (Danio rerio) to model muscle abnormalities due to defects in the subunits of DPMS. The three zebrafish ortholog subunits (encoded by the dpm1, dpm2 and dpm3 genes, respectively) showed high similarity to the human proteins, and their expression displayed localization in the midbrain/hindbrain area and somites. Antisense morpholino oligonucleotides targeting each subunit were used to transiently deplete the dpm genes. The resulting morphant embryos showed early death, muscle disorganization, low DPMS complex activity, and increased levels of apoptotic nuclei, together with hypoglycosylated α-DG in muscle fibers, thus recapitulating most of the characteristics seen in patients with mutations in DPMS. Our results in zebrafish suggest that DPMS plays a role in stabilizing muscle structures and in apoptotic cell death.

The aim of the present study was to evaluate the effects of three formulated diets: wheat gluten protein-based diets supplemented with LysGly dipeptide (PP) or free lisine and glycine (AA), and a control diet without lysine supplementation (CON), two commercial starters: Aller Futura (AF), Aglo Norse (AN) and frozen Chironomidae larvae (CH) for on the growth and digestive system development of rainbow trout. The experiment lasted 28 days and survival at the end of the experiment was the highest in the group fed AF (87.9 +/- 4.2%) and the lowest in the group fed the CON diet (74.0 +/- 4.3%). Fish fed the CON diet showed a significantly lower crude protein content, and the AA group had a significantly higher lipid level (P=0.05) in body carcass compared with the other groups. The highest density of intestinal goblet cells (P=0.05) was observed in fish fed the CON diet, while the lowest density was seen in the distal intestine in the AF group. The hepatocyte cytoplasm of all fish stored more glycogen than lipids. These results indicate that wheat gluten protein-based diets supplemented with dipeptide LysGly (PP) or free lysine and glycine (AA) supported growth, protein efficiency rate and intestine epithelium homeostasis. On the other hand, fish fed CON (lysine-deficient diet) showed lower survival and growth rate, and disturbances of intestine epithelium homeostasis, probably as a result of lysine deficiency.

The resistance demonstrated by many microorganisms towards conventional antibiotics has stimulated the interest in alternative antimicrobial agents and in novel approaches for prevention of infections. Silver, a natural braod-spectrum antimicrobial agent known since antiquity, has been widely employed in biomedical field due to its recognized antibacterial, antifungal and antiviral properties. In this work, antibacterial silver coatings were deposited on absorbable surgical sutures through the in situ photo-chemical deposition of silver clusters. Scanning electron microscopy (SEM), Energy dispersive X-ray spectroscopy (EDX) and thermo-gravimetric analysis (TGA) were performed in order to investigate the presence and distribution of the silver clusters on the substrate. The amounts of silver deposited and released by the silver treated sutures were calculated through Inductively Coupled Plasma-Mass Spectroscopy (ICP-MS), and the results were related to the biodegradation of the material. The microbiological properties and the potential cytotoxicity of the silver-treated sutures were investigated in relation with hydrolysis experiments, in order to determine the effect of the degradation on antibacterial properties and biocompatibility.

In this paper we describe the one-pot fabrication of hydroxyapatite (HA)-heparin composites by electrodeposition onto Ti substrates and their characterisation in terms of structure, morphology, heparin content and bioactivity. HA coatings are well known and widely applied osteointegration enhancers, but post-implant healing rate in dental applications is still suboptimal: e.g. coagulation control plays a key role and the incorporation of an anticoagulant is considered a highly desirable option. In this study, we have developed an improved, simple and robust growth procedure for single-phase, pure HA-heparin films of thickness 1/3 l m. HA-heparin, forming nanowires, has the ideal morphology for bone mineralisation. Staining assays revealed homogeneous incorporation of sizable amounts of heparin in the composite films. The bioactivities of the HA and HA-heparin coatings on Ti were compared by HeLa cell proliferation/viability tests and found to be enhanced by the presence of the anticoagulant.

Il compito della fisiologia animale è quello di descrivere, analizzare e comparare, qualitativamente e quantitativamente, le funzioni che caratterizzano il mondo animale, dagli organismi unicellulari che possiedono tutte le funzioni a quelli pluricellulari più complessi la cui integrità è garantita dalle funzioni di cellule specializzate e organizzate in tessuti e organi. Il libro inizia con la presentazione delle caratteristiche chimico-fisiche dei diversi ambienti terrestri, a cui fare riferimento per capire o giustificare la comparsa di un determinato adattamento che ha reso possibile la vita in condizioni apparentemente impossibili. Viene proposta una fisiologia animale integrata con le strategie adottate per l’adattamento all’ambiente di vita, che esamina attraverso strumenti moderni e sofisticati non solo le differenze fisiologiche tra gli organismi, ma anche i fini processi e i geni che sottendono alle differenze esistenti. Lo scopo di questo libro è di offrire agli studenti universitari che seguono vari corsi di Fisiologia fonti utili di informazioni sui sistemi fisiologici e spiegazioni chiare dei concetti fisiologici facendo riferimento agli animali (vertebrati e invertebrati) nel loro ambiente naturale, non escludendo principi e concetti legati ai vari processi fisiologici per poter capire e valutare l’origine dei vari processi e considerando argomenti di attualità come la percezione degli stimoli esterni, le strategie respiratorie (la fisiologia dell’immersione, i pigmenti respiratori) o le migrazioni. Per catturare e mantenere l’interesse degli studenti sono state inserite fotografie, figure e schemi originali per illustrare e soprattutto chiarire i vari aspetti discussi nel testo. Il risultato è un testo dai contenuti molto aggiornati, con approfondimenti che originano da ricerche scientifiche molto recenti.

Peptide transport and expression of SoLute Carrier 15 (SLC15) peptide transporters was assessed in rat thyroid tissue and a rat thyroid cell line (PC Cl3 cells). Peptide transport was studied by monitoring the uptake of the fluorophore-conjugated dipeptide beta-Ala-Lys-N(epsilon)-7-amino-4-methyl-coumarin-3-acetic acid (Ala-Lys-AMCA). Expression of SLC15-specific mRNA transcripts was analyzed by RT-PCR. Of the two SLC15 transporters expressed in thyroid follicular cells, namely PEPT2 (SLC15A2) and PHT1 (SLC15A4), only PEPT2 was involved in peptide transport at the plasma membrane, with PHT1 most likely being intracellular. Interestingly, at the mRNA level PEPT2 was up-regulated under TSH stimulation. These findings represent the first evidence that peptide transport occurs in thyroid follicular cells. SLC15 transporters could participate to recycling of peptides derived from extracellular and lysosomal thyroglobulin proteolysis, both essential steps for thyroid hormone synthesis.

Human bocavirus (HBoV) is a parvovirus isolated about a decade ago and found worldwide in both respiratory samples, mainly from early life and children of 6-24 mo of age with acute respiratory infection, and in stool samples, from patients with gastroenteritis. Since then, other viruses related to the first HBoV isolate (HBoV1), namely HBoV2, HBoV3 and HBoV4, have been detected principally in human faeces. HBoVs are small non-enveloped single-stranded DNA viruses of about 5300 nucleotides, consisting of three open reading frames encoding the first two the non-structural protein 1 (NS1) and nuclear phosphoprotein (NP1) and the third the viral capsid proteins 1 and 2 (VP1 and VP2). HBoV pathogenicity remains to be fully clarified mainly due to the lack of animal models for the difficulties in replicating the virus in in vitro cell cultures, and the fact that HBoV infection is frequently accompanied by at least another viral and/or bacterial respiratory and/or gastroenteric pathogen infection. Current diagnostic methods to support HBoV detection include polymerase chain reaction, real-time PCR, enzyme-linked immunosorbent assay and enzyme immunoassay using recombinant VP2 or virus-like particle capsid proteins, although sequence-independent amplification techniques combined with next-generation sequencing platforms promise rapid and simultaneous detection of the pathogens in the future. This review presents the current knowledge on HBoV genotypes with emphasis on taxonomy, phylogenetic relationship and genomic analysis, biology, epidemiology, pathogenesis and diagnostic methods. The emerging discussion on HBoVs as true pathogen or innocent bystander is also emphasized.

In tissue engineering field, the production of a porous resorbable matrix, termed scaffold, allows to host cells and guide them towards the synthesis of physiological tissue. Porous scaffolds provide mechanical stability and an initial framework for migrating cells and vascular infiltration. Sustained delivery of bioactive molecules at the defect site may be also particularly important for tissue regeneration. In this context, the goal of this work was the fabrication of highly porous collagen-based scaffolds incorporating uniformly dispersed poly(lactide-co-glycolide) (PLGA) microparticles as depots for the sustained and localized delivery of bioactive molecules. Collagen scaffolds loaded with different amounts of PLGA-microparticles were prepared by freeze-drying and crosslinking. The scaffolds microstructure was assessed to evaluate the spatial distribution of microparticles and the achieved pore size. The impact of the microparticles on the scaffolds stiffness was investigated through compression tests. Preliminarily, the cell-microparticles interactions were also evaluated by imaging of cell morphology in vitro, adopting a human derived epithelial cell model. The experimental findings showed that collagen scaffolds with different amounts of uniformly dispersed PLGA-microparticles were successfully produced. The microparticles did not negatively affect the scaffold porous structure, while acting as a mechanical reinforcement. Additionally, microparticles show high permissiveness to cell adhesion, and the interactions between microparticles and epithelial cell membranes did not interfere with the correct cells morphological differentiation. Such promising results suggest the potential of the developed scaffolds for tissue engineering applications.

Cisplatin, cis-diamminedichloroplatinum(II), is one of the most widely used antitumor drugs in cancer chemotherapy, as a critical component against a broad range of malignancies. Platinum anticancer drugs are generally known to target DNA, where they can bind to the N7 of purine bases. Cisplatin, as other bifunctional agents, is able to bind to adjacent purines, resulting in crosslink lesions believed to be responsible for the observed antitumor activity. N7-metalated purines, in some cases, seem to be characterized by a relevant antitumor activity. This has led us to hypothesize a cisplatin parallel mechanism of action: based on free platinated purines formation, directly in tissues, after drug administration. In order to evaluate this possible mechanism, as a key path to develop new drugs, we performed a series of experiments focused on platinated nucleobases cell and mitochondrial uptake and processing. For the first time cell uptake and mobility mechanisms, related to plasmatic cell and/or mitochondrial membrane crossing, has been studied. Moreover the possible insertion of metalated nucleobases into nuclear and/or mitochondrial new synthesized DNA/RNA, operated by DNA/RNA polymerases was evaluated. At this scope model metalated nucleosides/nucleotides, with nitrogen carrier ligands, have been synthesized, isolated and characterized. The possible development of new drugs based on this new rational drug design will be discussed.

Changes in the mitochondrial DNA (mtDNA) population, together with the expression of a set of genes involved in mtDNA replication and transcription and genes encoding for components of OxPhos complexes, were studied during zebrafish development from early embryo to larval stages. The mtDNA copy number, measured from 1h post-fertilization to the adult stage, significantly decreased over time, suggesting that mtDNA replication is not active in early zebrafish embryos and that, as in mammals, there occurs partition of the maternal mtDNA copies. Zebrafish genes involved in mtDNA replication (i.e. catalytic subunit of the mtDNA polymerase γ, mitochondrial deoxyribonucleoside kinase) are expressed late in embryo development, further supporting the notion that there is no replication of mtDNA in the early stages of zebrafish development. Notably, as from 4days post-fertilization, marked expression of "replication genes" was observed in the exocrine pancreas. Interestingly, the mtDNA helicase, also involved in mtDNA replication, was detected early in development, suggesting diverse regulation of this gene. On the other hand, zebrafish mtDNA transcription genes (i.e. mtDNA-directed RNA polymerase, mitochondrial transcription factor A) were ubiquitously expressed in the early stages of development, suggesting that mitochondrial transcription is already active before mtDNA replication. This hypothesis of early activation of mtDNA transcription fits in with the high early expression of structural OxPhos genes, suggesting that an active OxPhos system is necessary during early embryogenesis. As well as providing the first description of mtDNA distribution during zebrafish development, the present study also represents a step toward the use of Danio rerio as a model for investigation of mitochondrial metabolism and disease.

Atlantic salmon (Salmo salar L.) is one of the most economically important cultured fish and also a key model species in fish nutrition. During digestion, dietary proteins are enzymatically cleaved and a fraction of degradation products in the form of di- and tripeptides translocates from the intestinal lumen into the enterocyte via the Peptide Transporter 1 (PepT1). With this in mind, a full-length cDNA encoding the Atlantic salmon PepT1 (asPepT1) was cloned and functionally characterized. When overexpressed in Xenopus laevis oocytes, asPepT1 operated as a low-affinity/high-capacity transport system, and its maximal transport activity slightly increased as external proton concentration decreased (varying extracellular pH from 6.5 to 8.5). A total of 19 tested di- and tripeptides, some with acknowledged bioactive properties, some containing lysine, which is conditionally growth limiting in fish, were identified as well transported substrates, with affinities ranging between approximately 0.5 and approximately 1.5 mmol/L. Analysis of body tissue distribution showed the highest levels of asPepT1 mRNA in the digestive tract. In particular, asPepT1 mRNA was present in all segments after the stomach, with higher levels in the pyloric caeca and midgut region and lower levels in the hindgut. Depriving salmon of food for 6 d resulted in a approximately 70% reduction of intestinal PepT1 mRNA levels. asPepT1 will allow systematic in vitro analysis of transport of selected di- and tripeptides that may be generated in Atlantic salmon intestine during gastrointestinal transit. Also, asPepT1 will be useful as a marker to estimate protein absorption function along the intestine under various physiological and pathological conditions.

In this work, we assessed the capacity of RNA polymerases to use platinated ribonucleotides as substrates for RNA synthesis by testing the incorporation of the model compound [Pt(dien)(N7-5'-GTP)] (dien=diethylenetriamine; GTP=5'-guanosine triphosphate) into a natural RNA sequence. The yield of in vitro transcription operated by T7 RNA polymerase, on the LacZ (Escherichia coli gene encoding for β-galactosidase) sequence, decreases progressively with decreasing the concentration of natural GTP, in favor of the platinated nucleotide, [Pt(dien)(N7-5'-GTP)]. Comparison of the T7 RNA polymerase transcription activities for [Pt(dien)(N7-5'-GTP)] compound incorporation reaction test, with respect to the effect of a decreasing concentration of natural GTP, showed no major differences. A specific inhibitory effect of compound [Pt(dien)(N7-5'-GTP)] (which may pair the complementary base on the DNA strand, without being incorporated in the RNA by the T7 RNA polymerase) was evidenced. Our findings therefore suggest that RNA polymerases, unlike DNA polymerases, are unable to incorporate N7-platinated nucleotides into newly synthesized nucleic acids. In this respect, specifically designed N7-platinated nucleotides based compounds could be used in alternative to the classical platinum based drugs. This approach may offer a possible strategy to target specifically DNA, without affecting RNA, and is potentially able to better modulate pharmacological activity.

The dinoflagellates of the genus Ostreopsis Schmidt are toxic species involved in the occurrence of massive blooms. These dinoflagellates are known to produce palytoxin and ovatoxins, which are considered amongst the most poisonous phycotoxins in the world. In this work, aqueous and methanolic extracts of one Ostreopsis cf. ovata Ionian strain were tested on the human-derived HeLa cell line, and cytotoxicity, cytoskeletal rearrangement, and apoptosis induction were recorded by morphological and molecular analysis. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test revealed high toxicity for both extracts, which activity was inhibited by ouabain, which suggests the main involvement of palytoxin-like molecules in the observed toxic effects of this strain. Overall, the effects induced by O. cf. ovata on HeLa cells were dependent on the type of adopted extracts, being both toxic, but with the methanolic extract more toxic than the aqueous one and able to induce cell death by apoptosis. This process was evidenced by both phosphatidylserine exposition and upregulation of caspase-3 gene expression. The different effect of the two extracts on cell death is indicative of their different composition and/or activity. Our findings represent the starting step for the characterization of novel bioactive molecule

Protein digestion products are transported from the intestinal lumen into the enterocyte both in the form of free amino acids (AAs), by a large variety of brush border membrane AA transporters, and in the form of di/tripeptides, by a single brush border membrane transporter known as PEPtide Transporter 1 (PEPT1). Recent data indicate that, at least in teleost fish, PEPT1 plays a significant role in animal growth by operating, at the gastrointestinal level, as part of an integrated response network to food availability that directly supports body weight. Notably, PEPT1 responds to both fasting and refeeding and is involved in a phenomenon known as compensatory growth (a phase of accelerated growth when food levels are restored after a period of growth depression). In particular, PEPT1 expression decreases during fasting and increases during refeeding, which is the opposite of what observed so far in mammals and birds. These findings in teleost fish document, to our knowledge, for the first time in a vertebrate model, a direct correlation between the expression of an intestinal transporter, such as PEPT1, primarily involved in the uptake of dietary protein degradation products and animal growth.

Peptide transporters of the SLC15 family are classified by structure and function into PEPT1 (low‐affinity/high‐capacity) and PEPT2 (high‐affinity/low‐capacity) isoforms. Despite the differences in kinetics, both transporter isoforms are reckoned to transport essentially all possible di‐ and tripeptides. We here report that the fluorophore‐conjugated dipeptide derivatives β‐Ala‐Lys‐N‐7‐amino‐4‐methylcoumarin‐3‐acetic acid (β‐AK‐AMCA) and d‐Ala‐Lys‐N‐7‐amino‐4‐methylcoumarin‐3‐acetic acid (d‐AK‐AMCA) are transported by distinct PEPT isoforms in a species‐specific manner. Transport of the fluorophore peptides was studied (1) in vitro after heterologous expression in Xenopus oocytes of PEPT1 and PEPT2 isoforms from different vertebrate species and of PEPT1 and PEPT2 transporters from Caenorhabditis elegans by using electrophysiological and fluorescence methods and (2) in vivo in C. elegans by using fluorescence methods. Our results indicate that both substrates are transported by the vertebrate “renal‐type” and the C. elegans “intestinal‐type” peptide transporter only. A systematic analysis among species finds four predicted amino acid residues along the sequence that may account for the substrate uptake differences observed between the vertebrate PEPT1/nematode PEPT2 and the vertebrate PEPT2/nematode PEPT1 subtype. This selectivity on basis of isoforms and species may be helpful in better defining the structure–function determinants of the proteins of the SLC15 family.

PON 254/Ric. Potenziamento del “Centro Ricerche per la Salute dell’Uomo e dell’Ambiente” Cod. PONa3_00334;Consorzio Interuniversitario di Ricerca in Chimica dei Metalli nei Sistemi Biologici (CIRCMSB), Bari (Italy).

Adaptation of organisms to extreme environments requires proteins to work at thermodynamically unfavorable conditions. To adapt to subzero temperatures, proteins increase the flexibility of parts of, or even the whole, 3D structure to compensate for the lower thermal kinetic energy available at low temperatures. This may be achieved through single-site amino acid substitutions in regions of the protein that undergo large movements during the catalytic cycle, such as in enzymes or transporter proteins. Other strategies of cold adaptation involving changes in the primary amino acid sequence have not been documented yet. In Antarctic icefish (Chionodraco hamatus) peptide transporter 1 (PEPT1), the first transporter cloned from a vertebrate living at subzero temperatures, we came upon a unique principle of cold adaptation. A de novo domain composed of one to six repeats of seven amino acids (VDMSRKS), placed as an extra stretch in the cytosolic COOH-terminal region, contributed per se to cold adaptation. VDMSRKS was in a protein region uninvolved in transport activity and, notably, when transferred to the COOH terminus of a warm-adapted (rabbit) PEPT1, it conferred cold adaptation to the receiving protein. Overall, we provide a paradigm for protein cold adaptation that relies on insertion of a unique domain that confers greater affinity and maximal transport rates at low temperatures. Due to its ability to transfer a thermal trait, the VDMSRKS domain represents a useful tool for future cell biology or biotechnological applications.

Human genes for passive, ion-coupled transporters and exchangers are included in the so-called solute carrier (SLC) gene series, to date consisting of 52 families and 398 genes. Teleost fish genes for SLC proteins have also been described in the last two decades, and catalogued in preliminary SLC-like formin 50 families and at least 338 genes after systematic GenBank database mining (December 2010–March 2011).When the kinetic properties of the expressed proteins are studied in detail, teleost fish SLC transporters always reveal extraordinary ‘molecular diversity’ with respect to the mammalian counterparts, which reflects peculiar adaptation of the protein to the physiology of the species and/or to the environment where the species lives. In the case of the H+–oligopeptide transporter PEPT1(SLC15A1), comparative analysis of diverse teleost fish orthologs has shown that the protein may exhibit very eccentric properties in terms of pH dependence (e.g. the adaptation of zebrafish PEPT1 to alkaline pH), temperature dependence (e.g. the adaptation of icefish PEPT1 to sub-zero temperatures) and/or substrate specificity (e.g. the species-specificity of PEPT1 for the uptake of L-lysine-containing peptides). The revelation of such peculiarities is providing new contributions to the discussion on PEPT1 in both basic (e.g. molecular structure–function analyses) and applied research (e.g. optimizing diets to enhance growth of commercially valuable fish).

The present study evaluates the effect of protein source (dipeptides, free amino acids, and intact protein) on development and growth of Salmonid fish alevin. Specifically, we follow the expression of oligopeptide transporter protein PepT1 in the intestine of rainbow trout (Oncorhynchus mykiss). Fish were fed exogenously one of four diets: three formulated (lysyl-glycine dipeptide supplemented diet - PP, free lysine and glycine supplemented diet - AA, control diet with no lysine - CON) or commercial starter (Aller Futura - AF). Fish increased mean body weight 8 fold with PP- and AA-supplemented diets resulting in significantly higher weight gain than fish fed CON. Statistical analysis revealed a significant increase in relative PepT1 expression of fish fed experimental diets. Immunohistochemical staining with PepT1 antibody showed the presence of the transporter protein in the brush border membrane of the proximal intestinal enterocytes of fish from all experimental groups. Leptin immunoreactivity occurred not only in the gastric glands but also in proximal intestine and pyloric caeca of fish fed PP, AA and AF diets. Leptin immunoreactivity was also observed in hepatocyte cytoplasm and pancreatic acinar cells. Gastrin/CCK immunoreactive cells were present in the proximal intestine and pyloric caeca.

Common carp (Cyprinus carpio) of average body mass 0.07+/-0.02 g were fed three formulated diets: wheat gluten protein-based diet supplemented with Lys-Gly dipeptide (PP), wheat gluten protein-based diet supplemented with free lysine and glycine (AA), and a wheat gluten protein-based control diet without lysine supplementation (CON), frozen zooplankton (Z) (restricted diet), and a commercial starter food Aglo Norse (AN). After 4 weeks of experimental feeding, fish fed AN diet showed the highest body mass and length. Significantly lower mass occurred in groups fed PP, AA, CON, and Z. Fish fed CON diet showed the lowest intestinal folds and the highest number of mucous cells. Fish fed PP diet showed a significantly higher number of gastrin/cholecystokinin (CCK) positive cells. The diameter of lipid vacuoles in hepatocyte cytoplasm of fish fed formulated diets (PP, AA and CON) was significantly higher than in fish fed zooplankton (Z) and the commercial diet (AN). Hepatocytes of fish fed AA and CON showed a higher nucleus proliferation rate than in the other experimental groups. The quantitative analysis of the number of proliferating cell nuclear antigen (PCNA) and caspase-3(rabbit polyclonal antibody CPP-32)-positive cells showed that the highest proliferation rate was accompanied by the high apoptosis in the intestine of fish fed AA and CON. After 4 weeks of experimental feeding the highest relative expression of PepT1 gene was observed in fish fed PP diet, while the lowest expression occurred in fish fed CON. Feeding carp plant protein-based diet supplemented with Lys-Gly dipeptide (PP) had a beneficial influence on fish growth and metabolism in the digestive tract as compared to fish fed control diet without lysine supplementation (CON).

Human genes encoding passive transporters, ion-coupled transporters, and exchangers are all included in the so-called SoLute Carrier (SLC) gene series (the Human Genome Organization Gene Nomenclature Committee; http://www.genenames.org/), consisting of 51 families and at least 378 genes (http://www.bioparadigms.org). Ortholog genes encoding for transport proteins of the SLC series have comparatively been described in teleost fish, although their functional properties, in terms of kinetic parameters, substrate specificities, and inhibition patterns of the expressed transport proteins, have only sporadically been assessed in vitro. This chapter gives the latest updates (March 2011) for the SLC families and their members in teleost fish as well as relevant links to GenBank database and literature. By using a functional genomics approach, a list (version 1.0) of all currently known SLC families in teleost fish is provided in the form of SLC tables.

The exceptionally fast growth that fish experience after periods of fasting has been called “compensatory growth” or “catch up” growth. This phenomenon, reported in a wide range of fish species, has been studied in intensive aquaculture as a means of enhancing feed conversion efficiency, but the mechanisms implicated are complex and not yet fully understood. Our research focuses on identifying candidate genes whose expression contributes to the compensatory growth induced by refeeding in sea bass (Dicentrarchus labrax). As an alternative to genome sequencing for discovering genes in this teleost, we have successfully implemented a process for identifying unknown genes, assuming that a computational database resource contains orthologous sequences from phylogenetically related species. Following this similarity-driven molecular cloning and sequencing, we isolated several full-length sea bass cDNAs that encode for some physiologically relevant proteins in this species. We have then made use of the DNA sequence data to understand the function of these genes with the aim to identify valuable molecular biomarkers influenced, either positively or negatively, by dietary manipulations. Accordingly, in this chapter, we describe the utilization of molecular cloning and functional genomics in aquacultured sea bass to generate “transcriptome-focused” information, which may enable a better understanding of the transcriptional programs that underlie compensatory growth induced by refeeding.

The initial observation of peptide absorption in fish intestine dates back to 1981, when in rainbow trout (Oncorhynchus mykiss) the rate of intestinal absorption of the dipeptide glycylglycine (Gly-Gly) was compared in vivo to the rate of absorption of its component amino acid glycine (Gly). The chronicle of the identification of the underlying mechanisms that allow di- and tripeptide transport across the plasma membranes in fish starts in 1991, when the first evidence of peptide transport activity was reported in brush-border membrane vesicles (BBMV) of intestinal epithelial cells of Mozambique tilapia (Oreochromis mossambicus) by monitoring uptake of radiolabeled glycyl-L-phenylalanine (Gly-L-Phe). Since then, the existence of a carrier-mediated, H+-dependent transport of di- and tripeptides (H+/peptide cotransport) in the brush-border membrane of fish enterocytes has been confirmed in many teleost species by a variety of biochemical approaches, with the result of providing basic kinetics and substrate specificities of the transport activity. In 2003, the first peptide transporter from a teleost fish, i.e. the zebrafish (Danio rerio) PEPtide Transporter 1 (PEPT1), was cloned and functionally characterized in the Xenopus laevis oocyte expression system as a low-affinity/high-capacity system. PEPT1 is the protein in brush-border membranes responsible for translocation of intact di- and tripeptides released from dietary protein by luminal and membrane-bound proteases and peptidases. The transporter possesses affinities for the peptide substrates in the 0.1-10 mM range depending on the structure and physicochemical nature of the substrates. After the molecular and functional characterization of the zebrafish transporter, the interest on PEPT1 in teleost fish has grown and approaches for cloning and functional characterization of PEPT1 orthologues from other fish species, some of them of highest commercial value, are now underway. In this paper, we give a brief overview of the transport of di- and tripeptides in teleost fish intestine by recalling the bulk of biochemical, biophysical and physiological observations collected in the pre-cloning era and by recapitulating the more recent molecular and functional data.

Cisplatin, cis-diamminedichloroplatinum(II), is an effective antitumor drug known to exert its action by binding to the N7 of purine bases in nuclear DNA, consequently impairing replication, transcription and repair processes. Recently we have demonstrated, by the model complex [Pt(dien)(N7-5'-dGTP)] (dien = diethylenetriamine) the possible existence of an alternative mechanism for cisplatin antitumor activity, in which the metal is bonded to free purine bases, before incorporation in the newly synthesized DNA. This evidence suggests a new possible approach for the development of N7-platinated purines, as new antitumor drugs, for the treatment of cancer diseases. However, it is known that the efficacy of a new drug it is strictly related upon the transport across the cells plasma membrane barrier which strongly condition the drug pharmacokinetic and the targeted cells bioavailability. In particular in this study, we tested the possibility that N7-platinated purines could be transported into cells. At this scope we synthesized model Pt(II) coordination compounds, of the type [Pt(dien)(N7-G)] (G = Guanine derivative) and analysed the uptake of each single platinated nucleoside/nucleotide into Hela cells, by measuring Pt accumulation, by Inductively Coupled Plasma-Atomic Emission Spectrometry (ICP-AES). The quantitative analysis of Pt has provided a strong evidence for transport of the N7 platinated nucleoside [Pt(dien)(N7-dGuo)] into Hela cells (see figure). Moreover, the observed intracellular Pt accumulation was strictly sodiumdependent suggesting a carrier-mediated uptake for the [Pt(dien)(N7-dGuo)]. Overall, these results suggest that N7-platination does not compromise cellular uptake of nucleosides and may have important implications in the development of new potential nucleoside analogues in cancer treatment.