Best minimally modified antisense oligonucleotides according to cell nuclease activity.

Minimally modified oligonucleotides belong to the second-generation antisense class. They are phosphodiester oligonucleotides with a minimum of phosphorothioate linkages in order to be protected against serum and cellular exonucleases and endonucleases. They activate RNase H, have weak interactions with proteins, and have thus a better antisense efficiency. Two of them have been designed from an all-phosphorothioate antisense oligonucleotide directed against mdrl-expressing cells. They are protected against serum and cellular enzymatic degradation by the self-forming hairpin d(GCGAAGC) at their 3'-end and by judiciously located phosphorothioate residues, depending on the cellular composition in exonucleases or endonucleases. Besides their already demonstrated ability to cleave pyrimidine sites, endonucleases show some specificity for CpG sites. Their activity is hindered if specific sites are involved in secondary structure as hairpin.     

Histochemical localization of IGF-I and IGF-II mRNA in the rat between birth and adulthood

We describe the postnatal ontogeny and localization of insulin-like growth factors I and II (IGF-I and -II) in the rat. We have used oligodeoxyribonucleotide probes for in situ hybridization (hybridization histochemistry) and for Northern blotting. IGF-II mRNA is strongly expressed in liver, skeletal muscle, perichondrium, leptomeninges and choroid plexus of the newborn. Demonstrable levels fall dramatically in the liver at 18-20 days postnatally but persist for longer periods in muscle and remain undiminished throughout life in the pia/choroid plexus, indicating that different control mechanisms operate in these tissues. IGF-I mRNA is predominantly found in the liver. Its level in this organ rises well before levels of IGF-II fall. This suggests that distinct factors govern the expression of IGF-I and -II genes.     

Structure-activity study of phosphoramido acid esters as acetylcholinesterasf inhibitors

Phosphoramido acid esters (CH₃)₂NP(O)X(p-OC₆H₄-CH₃) (containing P-Cl (1), P-O (2), P-F (3), P-CN (5), and P-N (4,6) bonds, X for 2, 4 and 6 is OCH₃, (C₂H₅)₂N and morpholin) have been synthesized to investigate the structure-activity study of AChE enzyme inhibition, through the parameters logP, δ³¹P and IC₅₀. After their characterization by ³¹P, ³¹P{¹H}, ¹³C, ¹H NMR, IR and mass spectroscopy, the parameters logP and δ³¹P (³¹P chemical shift in NMR) were used to evaluated the lipophilicity and electronical properties. The ability of compounds to inhibit human AChE was predicted by PASS software (version 1.193), and experimentally evaluated by a modified Ellman's assay.     

Revisiting the use of Iprodione and Trichoderma in the integrated management of onion white rot

The biocontrol properties of Trichoderma species are well documented, but their effectiveness in antagonism of the problematic Sclerotium cepivorum, the causal agent of white rot in Allium species, appears limited with reports of significant control only relating to deliberately-mutated strains of Trichoderma. Our previous studies have indicated the possibility of using selected naturally-occurring strains of the antagonist in the suppression of other diseases; now in vitro and controlled environment in vivo studies have indicated that a degree of control of Onion White Rot is possible, and that the selected antagonist strains can be used in integrated treatments with Iprodione to good effect. The possible value of such treatments is considered in light of other approaches to the suppression of this continuing problem.     

Experimental evolution of the grain of metabolic specialization in yeast

Adaptation to any given environment may be accompanied by a cost in terms of reduced growth in the ancestral or some alternative environment. Ecologists explain the cost of adaptation through the concept of a trade‐off, by which gaining a new trait involves losing another trait. Two mechanisms have been invoked to explain the evolution of trade‐offs in ecological systems, mutational degradation, and functional interference. Mutational degradation occurs when a gene coding a specific trait is not under selection in the resident environment; therefore, it may be degraded through the accumulation of mutations that are neutral in the resident environment but deleterious in an alternative environment. Functional interference evolves if the gene or a set of genes have antagonistic effects in two or more ecologically different traits. Both mechanisms pertain to a situation where the selection and the alternative environments are ecologically different. To test this hypothesis, we conducted an experiment in which 12 experimental populations of wild yeast were each grown in a minimal medium supplemented with a single substrate. We chose 12 different carbon substrates that were metabolized through similar and different pathways in order to represent a wide range of ecological conditions. We found no evidence for trade‐offs between substrates on the same pathway. The indirect response of substrates on other pathways, however, was consistently negative, with little correlation between the direct and indirect responses. We conclude that the grain of specialization in this case is the metabolic pathway and that specialization appears to evolve through mutational degradation.     

Effect of lipid composition on incorporation of trastuzumab-PEG-lipid into nanoliposomes by post-insertion method: physicochemical and cellular characterization

Context: Anti-HER2 immunoliposomes are promising nanotechnology based systems for active targeting of breast tumors, which depends on the amount of incorporated antibody.

Objective/Aim: In this work, we investigated the possible effect of lipid composition on the incorporation of trastuzumab-PEG-PE micelles into nanoliposomes and on their subsequent specific cellular targeting.

Materials and methods: Trastuzumab (anti-HER2 monoclonal antibody) was monothiolated and conjugated to maleimide-PEG-PE micelles. Liposomes of different lipid compositions were prepared by the thin layer hydration. Trastuzumab-PEG-PE micelles were incorporated into the liposomes by the post-insertion method. The percentage of lipid mixing was determined based on fluorescence resonance energy transfer. Cellular binding and uptake of rhodamine-labeled immunoliposomes were studied in SKBR-3 (HER2⁺⁺⁺) and MCF-7 (HER2⁺) cells. Also, antitumor cell activity of the immunoliposomes was compared to free trastuzumab and the liposomes.

Results: The lipid mixing of trastuzumab-PEG-PE micelles depended on the liposome composition. The immunoliposomes containing DPPC, cholesterol and PEG-PE showed prominent lipid mixing. The lipid mixing was consistent with the cell binding results which showed an efficient and specific binding of the immunoliposomes to SKBR-3 cells. Antitumor cell activity of the immunoliposomes in SKBR-3, unlike MCF-7 cells, depended on the content of trastuzumab.

Discussion: Cholesterol and PEG-PE in the liposome composition are prerequisites for a successful lipid mixing due to their ability to facilitate fusion. The higher lipid mixing results in higher antibody incorporation and consequently higher targeted cell binding.

Conclusions: The lipid mixing depends on the liposome composition, which reflects targeted cell binding of the immunoliposomes.     

Evaluation of the neuroprotective effects of electromagnetic fields and coenzyme Q10 on hippocampal injury in mouse

Electromagnetic fields (EMFs) are reported to interfere with chemical reactions involving free radical production. Coenzyme Q₁₀ (CoQ10) is a strong antioxidant with some neuroprotective activities. The purpose of this study was to examine and compare the neuroprotective effects of EMF and CoQ10 in a mouse model of hippocampal injury. Hippocampal injury was induced in mature female mice (25–30 g), using an intraperitoneal injection of trimethyltin hydroxide (TMT; 2.5 mg/kg). The experimental groups were exposed to EMF at a frequency of 50 Hz and intensity of 5.9 mT for 7 hr daily over 1 week or treated with CoQ10 (10 mg/kg) for 2 weeks following TMT injection. A Morris water maze apparatus was used to assess learning and spatial memory. Nissl staining and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) tests were also performed for the histopathological analysis of the hippocampus. Antiapoptotic genes were studied, using the Western blot technique. The water maze test showed memory improvement following treatment with CoQ10 and coadministration of CoQ10 + EMF. The Nissl staining and TUNEL tests indicated a decline in necrotic and apoptotic cell count following treatment with CoQ10 and coadministration of CoQ10 + EMF. The Western blot study indicated the upregulation of antiapoptotic genes in treatment with CoQ10, as well as coadministration. Also, treatment with EMF had no significant effects on reducing damage induced by TMT in the hippocampus. According to the results, EMF had no significant neuroprotective effects in comparison with CoQ10 on hippocampal injury in mice. Nevertheless, coadministration of EMF and CoQ10 could improve the neuroprotective effects of CoQ10.     

Galectin-1 interacts with beta-1 subunit of integrin

Galectin-1, a beta-galactoside-binding dimeric lectin, is involved in adhesion, migration, and proliferation of vascular smooth muscle cells (SMC), the key steps in the development of atherosclerosis and restenosis. Here we investigated the molecular basis of the interactions between galectin-1 and SMCs. Galectin-1 modulated SMC attachment in a dose- and beta-galactoside-dependent manner. Direct binding of galectin-1 to beta1 integrin was detected by the immune precipitation of beta1 integrin after chemical cross-linking of 125I-labelled galectin-1 to the cell surface proteins. Galectin-1 transiently increased availability of beta1 integrins on the cell surface to antibodies against beta1 integrin. Incubation of SMCs with galectin-1 transiently increased the amount of the active form of beta1 integrin and tyrosine phosphorylation of two cytoskeleton-associated proteins; one of them coincided with focal adhesion kinase (FAK). Galectin-1 is likely to affect SMC adhesion by interacting with beta1 integrin on the cell surface of SMCs and inducing outside-in signalling.