A miniaturized nebulization catheter for improved gene delivery to the mouse lung

BACKGROUND: The available methods for administration of gene delivery systems to the lungs of small animals via nebulization have several drawbacks. These include lack of control over the delivered dose and a negative impact on the stability of the formulation. This paper describes a new nebulization catheter device for the administration of plasmid-based gene delivery systems (polyplexes) as aerosols to the mouse lung in vivo. METHODS: The physical stability of naked pDNA and polyplexes formulated with chitosan oligomers and PEI was examined following nebulization with the catheter device. We also examined the in vitro transfection efficiency of the polyplexes recovered after nebulization. Lung distribution and gene expression after administration of the selected gene delivery systems to the mouse lung were also investigated. RESULTS: In contrast to previously described nebulization methods, the structural integrity of the unprotected naked pDNA was maintained following nebulization by the catheter device, which indicates relatively mild nebulization conditions. In addition, the nebulization procedure did not affect the physical stability of the formulated polyplexes. Small volumes of the pDNA aerosol (10-20 microl) were delivered in a highly controlled and reproducible manner. The aerosol droplet size varied with the molecular weight of the polycations. Aerosol delivery via this method resulted in improved lung distribution of pDNA polyplexes and a six-fold increase in the efficiency of gene delivery in vivo over that seen with the commonly used intratracheal instillation method. CONCLUSION: The use of the nebulization catheter device provides a promising alternative for aerosol gene delivery to the mouse lung.     

Complete genome sequence of the obligate piezophilic hyperthermophilic archaeon Pyrococcus yayanosii CH1

Pyrococcus yayanosii CH1 is the first obligate piezophilic hyperthermophilic archaeon isolated from the deep-sea hydrothermal site Ashadze on the mid-Atlantic ridge at a depth of 4,100 m. This organism grows within a temperature range of 80 to 108°C and a hydrostatic pressure range of 20 to 120 MPa, with optima at 98°C and 52 MPa, respectively. Here, we report the complete genome sequence (1,716,817 bp, with a G+C content of 51.6%) of the type strain P. yayanosii CH1(T) (= JCM 16557). This genomic information reveals a systematic view of the piezoadaptation strategy and evolution scenario of metabolic pathways in Thermococcales.     

Protein-protein interactions in assembly of lipoic acid on the 2-oxoacid dehydrogenases of aerobic metabolism

Lipoic acid is a covalently attached cofactor essential for the activity of 2-oxoacid dehydrogenases and the glycine cleavage system. In the absence of lipoic acid modification, the dehydrogenases are inactive, and aerobic metabolism is blocked. In Escherichia coli, two pathways for the attachment of lipoic acid exist, a de novo biosynthetic pathway dependent on the activities of the LipB and LipA proteins and a lipoic acid scavenging pathway catalyzed by the LplA protein. LipB is responsible for octanoylation of the E2 components of 2-oxoacid dehydrogenases to provide the substrates of LipA, an S-adenosyl-L-methionine radical enzyme that inserts two sulfur atoms into the octanoyl moiety to give the active lipoylated dehydrogenase complexes. We report that the intact pyruvate and 2-oxoglutarate dehydrogenase complexes specifically copurify with both LipB and LipA. Proteomic, genetic, and dehydrogenase activity data indicate that all of the 2-oxoacid dehydrogenase components are present. In contrast, LplA, the lipoate protein ligase enzyme of lipoate salvage, shows no interaction with the 2-oxoacid dehydrogenases. The interaction is specific to the dehydrogenases in that the third lipoic acid-requiring enzyme of Escherichia coli, the glycine cleavage system H protein, does not copurify with either LipA or LipB. Studies of LipB interaction with engineered variants of the E2 subunit of 2-oxoglutarate dehydrogenase indicate that binding sites for LipB reside both in the lipoyl domain and catalytic core sequences. We also report that LipB forms a very tight, albeit noncovalent, complex with acyl carrier protein. These results indicate that lipoic acid is not only assembled on the dehydrogenase lipoyl domains but that the enzymes that catalyze the assembly are also present "on site."     

Differential signaling by adaptor molecules LRP1 and ShcA regulates adipogenesis by the insulin-like growth factor-1 receptor

The low density lipoprotein receptor-related protein (LRP1) is a transmembrane receptor that integrates multiple signaling pathways. Its cytoplasmic domain serves as docking sites for several adaptor proteins such as the Src homology 2/α-collagen (ShcA), which also binds to several tyrosine kinase receptors such as the insulin-like growth factor 1 (IGF-1) receptor. However, the physiological significance of the physical interaction between LRP1 and ShcA, and whether this interaction modifies tyrosine kinase receptor signaling, are still unknown. Here we report that LRP1 forms a complex with the IGF-1 receptor, and that LRP1 is required for ShcA to become sensitive to IGF-1 stimulation. Upon IGF-1 treatment, ShcA is tyrosine phosphorylated and translocates to the plasma membrane only in the presence of LRP1. This leads to the recruitment of the growth factor receptor-bound protein 2 (Grb2) to ShcA, and activation of the Ras/MAP kinase pathway. Conversely, in the absence of ShcA, IGF-1 signaling bifurcates toward the Akt/mammalian target of rapamycin pathway and accelerates adipocyte differentiation when cells are stimulated for adipogenesis. These results establish the LRP1-ShcA complex as an essential component in the IGF-1-regulated pathway for MAP kinase and Akt/mammalian target of rapamycin activation, and may help to understand the IGF-1 signaling shift from clonal expansion to growth-arrested cells and differentiation during adipogenesis.     

Effect of culture substrates on virulence of Beauveria bassiana (Ascomycota: Cordycipitaceae) conidia against the browntail moth,Euproctis chrysorrhoea (Lepidoptera: Lymantriidae)

We studied the effect of different solid substrates on virulence of two Beauveria bassiana isolates against the browntail moth, Euproctis chrysorrhoea (L.) (Lep.: Lymantriidae). Conidia produced on wheat grains, wheat flour, wheat bran, rice flour, rice bran, rice paddy, corn flour, millet, and Sabouraud's dextrose agar with 1% yeast extract (SDAY) as control were compared. There were significant differences among these substrates for their effects on the virulence of produced conidia. Applying 10⁷ conidia/mL of B. bassiana EUT105, produced on rice bran caused the highest (84.9%) and on rice flour, the lowest (57.6%) mortalities. Bioassay on fifth-instar larvae using aerial conidia harvested from wheat grains, rice paddy, and SDAY indicated that conidia from wheat grains had the highest virulence while those from rice paddy, the lowest.     

Synthesis of 3'-deoxy-3'-C-methyl nucleoside derivatives

2',3'-Dideoxy-3'-C-methyl nucleosides bearing the five naturally occurring nucleic acid bases were synthesized. Additionally, the 3'-deoxy-3'-C-methyl nucleoside analogues bearing 5-aminoimidazole-4-carboxamide as well as 1,2,4-triazole-3-carboxamide moieties were prepared. The synthesis of the corresponding 2',3'-dideoxy-3'-C-methyl triazole derivative was also accomplished. The dideoxynucleoside derivatives were prepared by radical deoxygenation from their 3'-deoxy-3'-C-methyl parent ribonucleosides. When evaluated for their antiviral activity in cell culture experiments, none of these compounds showed any significant antiviral activity.     

Recognition of hybrid peptidyl carrier proteins/acyl carrier proteins in nonribosomal peptide synthetase modules by the 4'-phosphopantetheinyl transferases AcpS and Sfp

The acyl carrier proteins (ACPs) of fatty acid synthase and polyketide synthase as well as peptidyl carrier proteins (PCPs) of nonribosomal peptide synthetases are modified by 4'-phosphopantetheinyl transferases from inactive apo-enzymes to their active holo forms by transferring the 4'-phosphopantetheinyl moiety of coenzyme A to a conserved serine residue of the carrier protein. 4'-Phosphopantetheinyl transferases have been classified into two types; the AcpS type accepts ACPs of fatty acid synthase and some ACPs of type II polyketide synthase as substrates, whereas the Sfp type exhibits an extraordinarily broad substrate specificity. Based on the previously published co-crystal structure of Bacillus subtilis AcpS and ACP that provided detailed information about the interacting residues of the two proteins, we designed a novel hybrid PCP by replacing the Bacillus brevis TycC3-PCP helix 2 with the corresponding helix of B. subtilis ACP that contains the interacting residues. This was performed for the PCP domain as a single protein as well as for the TycA-PCP domain within the nonribosomal peptide synthetase module TycA from B. brevis. Both resulting proteins, designated hybrid PCP (hPCP) and hybrid TycA (hTycA), were modified in vivo during heterologous expression in Escherichia coli (hPCP, 51%; hTycA, 75%) and in vitro with AcpS as well as Sfp to 100%. The designated hTycA module contains two other domains: an adenylation domain (activating phenylalanine to Phe-AMP and afterward transferring the Phe to the PCP domain) and an epimerization domain (converting the PCP-bound l-Phe to d-Phe). We show here that the modified PCP domain of hTycA communicates with the adenylation domain and that the co-factor of holo-hPCP is loaded with Phe. However, communication between the hybrid PCP and the epimerization domain seems to be disabled. Nevertheless, hTycA is recognized by the next proline-activating elongation module TycB1 in vitro, and the dipeptide is formed and released as diketopiperazine.     

Enzyme inhibition activities of Teucrium royleanum

The crude methanolic extract and chloroform, ethyl acetate and n-butanol fractions of Teucrium royleanum were examined as inhibitors of actylcholinesterase, butyrylcholinesterase, lipoxygenase and urease. A significant enzyme inhibition activity (52-83%) was shown by the crude methanolic extract and its fractions against acetylcholinesterase, while low to outstanding enzyme inhibitory activity was shown (19-93%) against butyrylcholinesterase. The crude methanolic extract and its various fractions demonstrated low activity against lipoxygenase and inactive against urease.     

Single and combined genotoxic and cytotoxic effects of two xenobiotics widely used in intensive aquaculture

Several chemicals are used in aquaculture to prevent or to treat disease outbreaks. These substances are mainly administered by two different routes: by prolonged immersion or by mixing into the diet. In the case of intensive aquaculture, the chemicals that are most frequently applied by immersion are formaldehyde (FA) 37% and oxytetracycline (OTC). The first is highly effective against most protozoa, as well as some of the most common parasites such as monogenetic trematodes. OTC presents a large spectrum of antibacterial activities and is used to treat systemic bacterial infections that affect fish. Under therapeutic use, FA (37%) is applied prophylactically at 200ml/m(3), whereas OTC is used curatively at 40g/m(3). The goal of the present study is to assess genotoxic and cytotoxic effects associated with exposure of the European sea bass (Dicentrarchus labrax) to FA37% and OTC under the same conditions as those applied in intensive aquaculture systems. To this end the micronucleus (MN) assay was applied in erythrocytes. Our results show that both tested chemicals present genotoxic and cytotoxic potential following a time-dependent pattern. Remarkably, the combined treatment induces a cumulative effect, which is particularly pronounced after 15 days of exposure. This suggests the critical hazards associated with exposure to FA and OTC when applied or released together.