Secondary metabolites,secretory structures and biological activity of water celery (Apium nodiflorum (L.) Lag.) growing in central Italy

Apium nodiflorum (L.) Lag. (water celery) is an hydrophytic plant forming dense submerged populations occurring along streams and rivers of Europe. In the present work we provided new insights into the phytochemistry and biology of A. nodiflorum. In particular, we studied the chemical profile of essential oil and polar extracts obtained from the flowering aerial parts of water celery growing in central Italy, together with the essential oil biological activities, namely antimicrobial, antioxidant and cytotoxicity on tumour cells. In addition, we correlated the productivity in secondary metabolites to the secreting structures through a detailed micromorphological study. The essential oil was dominated by two main chemotypes, characterized by myristicin and limonene, respectively. The oils showed significant toxicity on tumour cells, as evidenced by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, with IC₅₀ values in the range 3.8–15.9 μg mL^?1, together with inhibitory effects on Candida albicans (inhibition zones of 10–11 mm). HPLC-MS analysis showed the caffeoylquinic acids and quercetin-3-O-glucoside as the most abundant phenolic compounds. Ducts and vittae were the principal secretory structures of vegetative (leaves and stems) and reproductive (fruits) parts, respectively, storing mainly essential oil. Results of this work provide scientific evidences for the possible valorization and exploitation of water celery.     

Food habits of wolves in central Italy based on stomach and intestine analyses

We investigated wolfCanis lupus Linnaeus, 1758 food habits in central Italy by examining stomach and/or intestine contents of 59 individuals. Road accident and illegal kills were main causes of the wolves’ death. Ungulates represented the bulk of the diet (55% in frequency), and among them wild boar was the most important prey, followed by domestic Caprinae. Food items of domestic origin accounted for about 1/3 of all the diet. Diet composition did not vary between stomachs and intestines in spite of the higher degree of digestion of the intestines’ contents. The frequent detection of numerous larvae of Diptera and/or necrophagous Coleoptera, let suppose the consumption of already dead animals, and suggests a general underestimate of the wolf’s scavenging behaviour in previous studies based on scat analyses.     

Phospholipase C-gamma contains introns shared by src homology 2 domains in many unrelated proteins

Many proteins with novel functions were created by exon shuffling around the time of the metazoan radiation. Phospholipase C-gamma (PLC-gamma) is typical of proteins that appeared at this time, containing several different modules that probably originated elsewhere. To gain insight into both PLC-gamma evolution and structure-function relationships within the Drosophila PLC-gamma encoded by small wing (sl), we cloned and sequenced the PLC-gamma homologs from Drosophila pseudoobscura and D. virilis and compared their gene structure and predicted amino acid sequences with PLC-gamma homologs in other animals. PLC-gamma has been well conserved throughout, although structural differences suggest that the role of tyrosine phosphorylation in enzyme activation differs between vertebrates and invertebrates. Comparison of intron positions demonstrates that extensive intron loss has occurred during invertebrate evolution and also reveals the presence of conserved introns in both the N- and C-terminal PLC-gamma SH2 domains that are present in SH2 domains in many other genes. These and other conserved SH2 introns suggest that the SH2 domains in PLC-gamma are derived from an ancestral domain that was shuffled not only into PLC-gamma, but also into many other unrelated genes during animal evolution.     

A novel autoregulatory loop between the Gcn2-Atf4 pathway and L-Proline metabolism controls stem cell identity

Increasing evidence indicates that metabolism is implicated in the control of stem cell identity. Here, we demonstrate that embryonic stem cell (ESC) behaviour relies on a feedback loop that involves the non-essential amino acid L-Proline (L-Pro) in the modulation of the Gcn2-Eif2α-Atf4 amino acid starvation response (AAR) pathway that in turn regulates L-Pro biosynthesis. This regulatory loop generates a highly specific intrinsic shortage of L-Pro that restricts proliferation of tightly packed domed-like ESC colonies and safeguards ESC identity. Indeed, alleviation of this nutrient stress condition by exogenously provided L-Pro induces proliferation and modifies the ESC phenotypic and molecular identity towards that of mesenchymal-like, invasive pluripotent stem cells. Either pharmacological inhibition of the prolyl-tRNA synthetase by halofuginone or forced expression of Atf4 antagonises the effects of exogenous L-Pro. Our data provide unprecedented evidence that L-Pro metabolism and the nutrient stress response are functionally integrated to maintain ESC identity.Naturally occurring amino acids are emerging as key players in the regulation of the phenotypic plasticity of stem cells.^{1, 2, 3, 4, 5} Indeed, exogenously provided threonine and methionine, two essential amino acids (EAAs), regulate self-renewal and differentiation of pluripotent stem cells.² Moreover, exogenously provided L-Proline (L-Pro), a non-essential amino acid (NEAA), induces mouse ESCs towards an embryonic stem cell-to-mesenchymal-like transition (esMT) that converts compact, adherent ESCs into mesenchymal-like spindle-shaped, highly invasive and metastatic pluripotent stem cells.⁴ This fully reversible process resembles the epithelial-to-mesenchymal transition (EMT), which is essential for normal development and contributes to pathological cancer progression.^{6, 7, 8} Interestingly, the Aldh18a1 gene is specifically induced in and marks the Primitive Endoderm (PrE) in the time window when the pluripotent epiblast precursors are specified within the inner cell mass (ICM) of the blastocyst.⁹ Since the Aldh18a1 enzyme catalyses the first and rate-limiting step of L-Pro biosynthesis, these findings suggest that L-Pro metabolism may regulate cell lineage segregation in early mammalian embryos. Despite its relevance, the molecular mechanisms underlying L-Pro control of stem cell identity remain largely unknown. This prompted us to investigate the early molecular events regulated by exogenously provided L-Pro in mouse ESCs.     

PED/PEA-15 controls fibroblast motility and wound closure by ERK1/2-dependent mechanisms

Cell migration is dependent on the control of signaling events that play significant roles in creating contractile force and in contributing to wound closure. We evaluated wound closure in fibroblasts from mice overexpressing (TgPED) or lacking ped/pea-15 (KO), a gene overexpressed in patients with type 2 diabetes. Cultured skin fibroblasts isolated from TgPED mice showed a significant reduction in the ability to recolonize wounded area during scratch assay, compared to control fibroblasts. This difference was observed both in the absence and in the presence of mytomicin C, an inhibitor of mitosis. In time-lapse experiments, TgPED fibroblasts displayed about twofold lower velocity and diffusion coefficient, as compared to controls. These changes were accompanied by reduced spreading and decreased formation of stress fibers and focal adhesion plaques. At the molecular level, TgPED fibroblasts displayed decreased RhoA activation and increased abundance of phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2). Inhibition of ERK1/2 activity by PD98059 restored RhoA activation, cytoskeleton organization and cell motility, and almost completely rescued wound closure of TgPED fibroblasts. Interestingly, skin fibroblasts isolated from KO mice displayed an increased wound closure ability. In vivo, healing of dorsal wounds was delayed in TgPED and accelerated in KO mice. Thus, PED/PEA-15 may affect fibroblast motility by a mechanism, at least in part, mediated by ERK1/2.     

Macrolide resistance genotypes and phenotypes among erythromycin-resistant clinical isolates of Staphylococcus aureus and coagulase-negative staphylococci, Italy

One hundred macrolide-resistant staphylococcal isolates from clinically relevant infections in Italy during a 19-month period were studied. Four distinct resistance phenotypes were observed using the triple-disk induction test (erythromycin, clindamycin, telithromycin): the cMLS_B phenotype (24 isolates); the iMLS_B phenotype (41 isolates); the MS phenotype (three isolates); and the iMTS phenotype (erythromycin-induced telithromycin resistance) (32 isolates). ermC and ermA genes predominated within erythromycin-resistant Staphylococcus aureus isolates with iMLS_B phenotype and cMLS_B phenotype, respectively. Among erythromycin-resistant CoNS isolates, half of the strains showed the iMTS or MS/ msrA association, and ermC gene predominated among isolates with MLS_B phenotype. By pulsed-field gel electrophoresis, high genetic heterogeneity was observed among the isolates studied. Both independent acquisition of macrolide resistance genes and spread of specific resistant clones were observed. Association between certain clonal types and specific types of infection could be detected. To our knowledge, this is the first report on characterization of erythromycin-resistant staphylococci in Italy.     

Primula spectabilis Tratt. aerial parts: morphology, volatile compounds and flavonoids

The vacuolar and epicuticular flavonoids and the volatiles of the leaves and parts of flower of P. spectabilis Tratt., an endemic species in the Italian Oriental Alps, were investigated. From a MeOH extract of the leaves two flavone glycosides, 8-C-β-glucopyranosylluteolin 7-O-α-arabinofuranoside (1) and 6-C-α-arabinofuranosylapigenin (2) were isolated, in addition to a flavone and three flavonols already known from species of Primula. From an EtOH extract of leaf exudates, 7,3',4'-tri-O-methylquercetin was obtained. The structures were elucidated on the basis of their 1D 1H- and 13C NMR data and 2D NMR techniques, as well as of HPLC-MS. The volatiles emitted by the leaves were mainly constituted by non-terpene derivatives, followed by comparable proportions of hemiterpens, oxygenated monoterpenes and sesquiterpene hydrocarbons. In flowers, monoterpene hydrocarbons were the most represented chemical class followed by non-terpene derivatives. Different proportions of compounds were found when individual parts of flowers were examined separately; calyx produced a greater proportion (approx. 49.5%) of non-terpenes as its volatile metabolites. P. spectabilis has glandular trichomes in the hyaline margins of the epidermal depressions, distributed on the adaxial leaf blade. Glandular hairs were also present on the corolla. Correlations of phytochemical data with the morphological features of leaf, flower and glandular hair are discussed, and a hypothesis is proposed on the ecological roles of the flavonoids and volatile compounds on the general fitness of the species and cross-pollination strategies.     

Isolation of the human immunodeficiency virus (HIV) from the cerebro-spinal fluid

The authors describe a simple and available technique for HIV isolation from cerebrospinal fluid and report preliminary results obtained. Exposed data indicate that neurological involvement occurs early in the natural history of HIV infection and that the virus may be recovered in CSF at all stages of the disease, regardless of immunological conditions of patients. Virological evaluation of CSF may be important in understanding pathogenetic aspects of HIV infection and in clinical management of infected patients.     

Correlation of viral replication activity, the clinical phase of the disease and immunologic deterioration in HIV infection

The authors have studied the relationship among HIV replicative activity, expressed by HIV isolation from peripheral blood lymphocytes and p24 antigenemia detection, and the evolution of infection, in terms of stage of disease and T4 cells absolute number, in a group of 120 infected subjects. Data presented indicate that the expression of viral gene products is related both to clinical and immunological deterioration and suggest that viral replication plays a central role in the progression of the disease.     

Survival of Trichinella muscle larvae in frozen wolf tissue in Italy

Living larvae were collected in central Italy from the masseters of a wolf frozen for 6 mo. These parasites were identified as a southern Palearctic species (Trichinella sp. 3) through gene-enzyme analysis. At the second passage in laboratory mice, the muscle larvae were unable to survive at - 10 C for 5 days. Biological and epidemiological implications are discussed.