Physicochemical and biological study of selected hydrophobic polyethylenimine-based polycationic liposomes and their complexes with DNA

Non-viral gene therapy is based on the development of efficient and safe gene carrier systems able to transfer DNA into cells. Polyethylenimine (PEI), the most promising non-viral vector, with its high cationic-charge-density potential is able (1) to compact DNA in complexes (polyplexes) smaller than those formed by liposomes (lipoplexes) and (2) to destabilize the endosomal membrane by a 'proton sponge' effect. Several PEI's hydrophobic modifications were reported in the last several years but in some cases a reduced transfection efficiency was observed. The mechanism underlying this phenomenon is not well understood so far. In order to extensively investigate these mechanisms, we reported a physicochemical and biological study of selected hydrophobic PEI's derivatives grafted with chains of different length and percentages of substitution able to form vesicles (polycationic liposomes) and to bind DNA. Their properties were studied by means of dynamic light scattering, freeze-fracture microscopy, potentiometric titrations, gel retardation assays, polyanion exchange reactions, toxicity assays, in vitro transfections, and fluorescence microscopy. Our results indicate that even if polyplexes are able to pass through the cellular membrane, the stability of PEI's hydrophobic polyplexes likely explain their different transfection efficiency in vitro.     

Structural characterization of the O-chain polysaccharide from an environmentally beneficial bacterium Pseudomonas chlororaphis subsp. aureofaciens strain M71

Pseudomonas chlororaphis subsp. aureofaciens strain M71 was isolated from the root of a tomato plant and it was able to control in vivo Fusarium oxysporum f. sp. radicis-lycopersici responsible for the tomato crown and root rot. Recently, strain M71 was evaluated even for its efficacy in controlling Seiridium cardinale, the causal agent of bark canker of common cypress (Cupressus sempervirens L.). Strain M71 ability to persist on the tomato rhizosphere and on the aerial part of cypress plants could be related to the nature of the lipopolysaccharides (LPS) present on the outer membrane and in particular to the O-specific polysaccharide.A neutral O-specific polysaccharide was obtained by mild acid hydrolysis of the lipopolysaccharide from P. chlororaphis subsp. aureofaciens strain M71. By means of compositional analyses and NMR spectroscopy, the chemical repeating unit of the polymer was identified as the following linear trisaccharide.     

Synthesis and biological activity of a novel class nicotinic acetylcholine receptors (nAChRs) ligands structurally related to anatoxin-a

The introduction of the isoxazole ring as bioisosteric replacement of the acetyl group of anatoxin-a led to a new series of derivatives binding to nicotinic acetylcholine receptors. Bulkier substitutions than methyl at the 3 position of isoxazole were shown to be detrimental for the activity. The binding potency of the most interesting compounds with α1, α7 and α3β4 receptor subtypes, was, anyway, only at micromolar level. Moreover, differently from known derivatives with pyridine, isoxazole condensed to azabicyclo ring led to no activity.     

Kinetic and anion inhibition studies of a β-carbonic anhydrase (FbiCA 1) from the C4 plant Flaveria bidentis

Several β-carbonic anhydrases (CAs, EC 4.2.1.1) are present in all land plants examined thus far. Here we report the first detailed biochemical characterization of one such isoform, FbiCA 1, from the C₄ plant Flaveria bidentis, which was cloned, purified and characterized as recombinant protein. FbiCA 1 has an interesting CO₂ hydrase catalytic activity (k_cat of 1.2 × 10⁵ and k_cat/K_m of 7.5 × 10⁶ M^?1 × s^?1) and was moderately inhibited by most simple/complex inorganic anions. Potent FbiCA 1 inhibitors were also detected, such as trithiocarbonate, diethyldithiocarbamate, sulfamide, sulfamic acid, phenylboronic acid and phenylarsonic acid (K_Is in the range of 4–60 μM). Such inhibitors may be used as tools to better understand the role of various β-CA isoforms in photosynthesis.     

Antiendotoxin activity of protegrin analog IB-367 alone or in combination with piperacillin in different animal models of septic shock

The therapeutic efficacy of protegrin peptide IB-367 was investigated in three rat models of septic shock: (i) rats injected intraperitoneally with 1mg Escherichia coli 0111:B4 lipopolysaccharide, (ii) rats given an intraperitoneal injection of 2 X 10(10) CFU of E. coli ATCC 25922, and (iii) rats in which intra-abdominal sepsis was induced via cecal ligation and puncture. All animals were randomized to receive parenterally isotonic sodium chloride solution, 1mg/kg of IB-367, 60mg/kg piperacillin and 1mg/kg of IB-367 plus 60mg/kg piperacillin. The peptide demonstrated lower level of antimicrobial activity than piperacillin, nevertheless it exhibited the dual properties of antimicrobial and antiendotoxin agent. Finally IB-367 and piperacillin association showed to be the most effective therapeutic approach.     

Secondary metabolites from the aerial parts of Salvia palaestina Bentham

Three sesterterpenes (1-3), one triterpene (4) and five diterpenes (5-9) were isolated from the aerial parts of Salvia palaestina Bentham (Lamiaceae), together with two sesquiterpenes, 10 known diterpenes, three triterpenes, and rosmarinic acid. Their structural elucidation was accomplished by extensive spectroscopic methods including 1D ((1)H, (13)C, (13)C DEPT, TOCSY, NOESY) and 2D NMR experiments (DQF-COSY, HSQC, HMBC, ROESY) as well as ESIMS analysis and chemical analysis.     

Cannabidiol reduces Aβ-induced neuroinflammation and promotes hippocampal neurogenesis through PPARγ involvement

Peroxisome proliferator-activated receptor-γ (PPARγ) has been reported to be involved in the etiology of pathological features of Alzheimer's disease (AD). Cannabidiol (CBD), a Cannabis derivative devoid of psychomimetic effects, has attracted much attention because of its promising neuroprotective properties in rat AD models, even though the mechanism responsible for such actions remains unknown. This study was aimed at exploring whether CBD effects could be subordinate to its activity at PPARγ, which has been recently indicated as its putative binding site. CBD actions on β-amyloid-induced neurotoxicity in rat AD models, either in presence or absence of PPAR antagonists were investigated. Results showed that the blockade of PPARγ was able to significantly blunt CBD effects on reactive gliosis and subsequently on neuronal damage. Moreover, due to its interaction at PPARγ, CBD was observed to stimulate hippocampal neurogenesis. All these findings report the inescapable role of this receptor in mediating CBD actions, here reported.     

Growth hormone and ghrelin secretion in severely obese women before and after bariatric surgery

Objective: The objective was to evaluate ghrelin and growth hormone (GH) interactions and responses to a growth hormone‐releasing hormone (GHRH)/arginine test in severe obesity before and after surgically‐induced weight loss. Research Methods and Procedures: Our study population included 11 severely obese women 39 ± 12 years of age, with a mean BMI of 48.6 ± 2.4 kg/m², re‐studied in a phase of stabilized body weight, with a BMI of 33.4 ± 1.2 kg/m², 18 months after having successfully undergone biliopancreatic diversion (BPD). A GHRH/arginine test was performed before and 18 months after BPD to evaluate ghrelin and GH interactions. Active ghrelin, measured by radioimmunoassay (RIA), and GH, measured by chemiluminescence assay, were assayed before and after the GHRH/arginine test. Results: Fasting serum GH levels and GH area under the curve (AUC) significantly increased from 0.2 ± 0.05 ng/mL to 1 ± 0.3 ng/mL (p < 0.05) and from 514.76 ± 98.7 ng/mL for 120 minutes to 1957.3 ± 665.1 ng/mL for 120 minutes after bariatric surgery (p < 0.05), respectively. Although no significant change in fasting ghrelin levels was observed (573 ± 77.9 before BPD vs. 574.1 ± 32.7 after BPD), ghrelin AUC significantly increased from ?3253.9 ± 2180.9 pg/mL for 120 minutes to 1142.3 ± 916.4 pg/mL for 120 minutes after BPD (p < 0.05). Fasting serum insulin‐like growth factor (IGF)‐1 concentration did not change significantly (133.6 ± 9.9 ng/mL before vs. 153.3 ± 25.2 ng/mL after BPD). Discussion: Our study demonstrates that the mechanisms involved in ghrelin and GH secretion after the secretagogue stimulus (GHRH/arginine) are consistent with patterns observed in other populations.     

c-Jun N-terminal kinase upregulation as a key event in the proapoptotic interaction between transforming growth factor-beta1 and 4-hydroxynonenal in colon mucosa

Cells of colonic mucosa are sensitive to the Smad-mediated growth-inhibitory effect of transforming growth factor-beta1 (TGF-beta1). Another important cell growth inhibitor is the polyunsaturated lipid peroxidation end product, 4-hydroxynonenal (HNE), which triggers apoptosis through c-Jun N-terminal kinase (JNK) activation. Interestingly, a close association between TGF-beta1 and HNE was found in the progression of human colon cancer, with concentration of both molecules inversely related to the malignancy. We investigated the cross talk between Smads and JNK signal transduction pathways in inducing apoptosis. To this purpose TGF-beta1 and HNE were added singly or in combination to CaCo-2 human colon adenocarcinoma cells. The cotreatment induced a marked enhancement of apoptosis and of JNK and Smad4 activities much more than either individual molecule. Cell preincubation with the JNK inhibitor SP600125 significantly prevented JNK and Smad4 enhancement and, subsequently, the cooperative proapoptotic effect was abolished. The primary role of JNK activity in TGF-beta1/HNE cooperative signaling was fully confirmed in a second set of experiments by using JNKi I, a more selective kinase inhibitor. Hence, in tumor cells becoming resistant to TGF-beta1-mediated growth inhibition, increased induction of the remaining TGF-beta1 pathways by interaction with other antiproliferative molecules, such as HNE, could help in inhibiting tumor growth.