Preimplantation diagnosis for neurofibromatosis

Preimplantation genetic diagnosis (PGD) has recently been performed for inherited cancer predisposition determined by p53 tumour suppressor gene mutations, suggesting the usefulness of PGD for late onset disorders with genetic predisposition, including those caused by the germline mutations of other tumour suppressor genes. Here PGD was performed for two couples, one at risk for producing a child with maternally derived neurofibromatosis type I (NF1), and the other with paternally derived neurofibromatosis type II (NF2). The procedure involved a standard IVF protocol, combined with testing of oocytes or embryos prior to their transfer back to the patients. Maternal mutation Trp-->Ter (TGG-->TGA) in exon 29 of the NF1 gene was tested by sequential PCR analysis of the first and second polar bodies, and paternal L141P mutation in exon 4 of the NF2 gene by embryo biopsy at the cleavage stage. In both cases, multiplex nested PCR was applied, involving NF1 and NF2 mutation analysis simultaneously with the 3 and 2 linked markers, respectively. Of 57 oocytes tested in four PGD cycles for NF1 mutation, 26 mutation-free oocytes were detected, from which eight were preselected for transfer, two in each cycle. These produced two clinical pregnancies, one confirmed to be mutation free by chorionic villus sampling but ending in a stillbirth, and the other still ongoing. Of 18 embryos analysed in a cycle performed for NF2 mutation, eight mutation-free embryos were detected, three of which were transferred back to the patient, resulting in a singleton pregnancy and the birth of a mutation-free child. This suggests that PGD is a useful approach for avoiding the birth of children with inherited cancer predisposition, determined by NF1 and NF2 gene mutations.     

Requirement of Gbetagamma and c-Src in D2 dopamine receptor-mediated nuclear factor-kappaB activation

The D2 dopamine receptor (D2R) was examined for its ability to mediate nuclear factor-kappaB (NF-kappaB) activation through G proteins. Stimulation of D2R-transfected HeLa cells with its agonist quinpirole induced the expression of a NF-kappaB luciferase reporter and formation of NF-kappaB-DNA complex. This response was blocked by pertussis toxin, and by the Gbetagamma scavengers transducin and beta-adrenergic receptor kinase 1 carboxyl-terminal fragment. Unlike Gi-coupled chemoattractant receptors, D2R activated NF-kappaB without an increase in phospholipase C-beta activity, and the response was only slightly affected by the phosphoinositide 3-kinase inhibitor 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002). In contrast, treatment with genistein and 4-amino-1-tert-butyl-3-(p-methylphenyl)pyrazolo[3,4-d] pyrimidine abolished the induced NF-kappaB activation, suggesting involvement of protein tyrosine kinases. Activation of D2R led to phosphorylation of c-Src at Tyr-418, and expression of a kinase-deficient c-Src inhibited D2R-mediated NF-kappaB activation. The D2R-mediated NF-kappaB activation was not dependent on epidermal growth factor (EGF) receptor transactivation since 4-(3'-chloroanilino)-6,7-dimethoxyquinazoline (AG1478), an EGF receptor-selective tyrphostin used at 1 microM, blocked EGF-induced NF-kappaB activation but not the quinpirole-induced response. In addition, the D2R-mediated NF-kappaB activation was enhanced by over-expression of beta-arrestin 1. These results suggest that D2R-mediated NF-kappaB activation requires Gbetagamma and c-Src, and possibly involves beta-arrestin 1.     

Discovery of distinct protein profiles specific for lung tumors and pre-malignant lung lesions by SELDI mass spectrometry

Objectives: Early lung cancer detection and treatment remain a challenge. The efficacy of surface-enhanced laser desorption/ionization (SELDI) technology in lung cancer detection, has not been defined. This study identifies specific protein peak patterns in malignant lung tumors, and in pre-malignant airways epithelium showing neoplastic transformation. Methods: Lung tumor specimens taken from patients participating in a lung cancer screening study (H. Lee Moffitt Cancer Center, Tampa, FL) were laser capture microdissected to obtain pure cell populations from frozen sections of normal lung, atypical adenomatous hyperplasia (AAH) and malignant tumors. SELDI mass spectrometry was used to generate protein profiles in each epithelial cell type. Results: SELDI mass spectroscopy is highly reproducible in detecting lung tumor-specific protein profiles. Three peaks at 17–23 kDa mass range from tumor cells showed markedly increased compared with normal cells. The peak at 17 250 Da was not detected in any of the normal cells. This peak appeared to be present at low levels in the atypical cell samples. Conclusions: This study demonstrates the feasibility of detecting “malignant” protein signatures from lung tumor and pre-malignant pulmonary epithelium using SELDI mass spectrometry. Although additional study is necessary to validate these patterns as unique diagnostic tools, these “malignant” protein signatures lend themselves to identification of populations at high-risk for lung cancer and for monitoring response to lung cancer chemopreventive agents.     

Radiation enhances the anti-tumor effects of vaccinia-p53 gene therapy in glioma

The overall goal of this study was to analyze the effect and mechanism of radiation in combination with vaccinia viruses (VV) carrying the p53 gene against glioma. Comparison of two alternative treatments of cultured C6 (p53(+)) and 9L (p53(-)) rat glioma cells showed significantly reduced survival for both cell lines, especially 9L, when radiation was applied prior to virus versus radiation alone. High p53 protein expression mediated by VV-TK-p53 was measured in infected cells. Single modality treatment of C6 cells with psoralen and UV (PUV)-inactivated VV-TK-p53 (PUV-VV-TK-53) or radiation significantly decreased survival compared with PUV-inactivated L-15 (PUV-L-15) control virus. However, no difference was observed between radiation and combination treatments of C6 cells. In contrast, radiation followed by PUV-VV-TK-53 resulted in dramatic reduction of 9L cell viability, compared to single modality treatment. Flow cytometry analysis of Annexin-V-stained 9L cells showed that radiation and PUV-VV-TK-53 caused a significant decrease in live cells (17.2%) as compared to other treatments and control (61.6-98.3%). Apoptosis was observed in 37.2% of cells, while the range was 0.7-7.8% in other treatment groups; maximal p53 level was measured on day 7 post-infection. In athymic mice bearing C6 tumors, VV-TK-53 plus radiation in both single and multiple therapies resulted in significantly smaller tumors by day 30 compared to the agents given only once. Immunohistochemical analysis of tumor sections demonstrated p53 protein expression over 20 days after VV-TK-53 treatment. Analysis of blood and spleen cells of mice given multiple combination treatments showed significant splenomegaly, leukocytosis, and increased DNA synthesis and response to mitogen. Multiple combination treatments were also associated with significantly elevated natural killer and B cells in the spleen. There were no overt toxicities, although depression in red blood cell and thrombocyte parameters was noted. Collectively, the data demonstrate that radiation significantly improves the efficacy of VV-mediated tumor suppressor p53 therapy and may be a promising strategy for glioma treatment. Furthermore, the results support the conclusion that the mechanisms underlying the enhanced anti-tumor effect of combination treatment include apoptosis/necrosis and upregulation of innate immune defenses.     

Amphiphilic poly-N-vinylpyrrolidones: synthesis, properties and liposome surface modification

Certain amphiphilic water-soluble polymers including amphiphilic derivatives of polyvinyl pyrrolidone (PVP) were found to be efficient steric protectors for liposomes in vivo. In this study, we have tried to develop synthetic pathways for preparing amphiphilic PVP and to investigate the influence of the hydrophilic/hydrophobic blocks on some properties of resulting polymers and polymer-coated liposomes. To prepare amphiphilic PVP with the end stearyl (S) or palmityl (P) residues, amino- and carboxy-terminated PVP derivatives were first synthesized by the free-radical polymerization of vinyl pyrrolidone in the presence of amino- or carboxy-mercaptans as chain transfer agents, and then modified by interaction of amino-PVP with stearoyl chloride or palmitoyl chloride, or by dicyclohexyl carbodiimide coupling of stearylamine with carboxy-PVP. ESR-spectra of the hydrophobic spin-probe, nitroxyl radical N-oxyl-2-hexyl-2-(10-methoxycarbonyl)decyl-4,4'-dimethyl oxazoline, in the presence of amphiphilic PVP demonstrated good accessibility of terminal P- and S-groups for the interaction with other hydrophobic ligands. Spontaneous micellization and low CMC values (in a low micromolar range) were found for amphiphilic PVP derivatives using the pyrene method. In general, S-PVP forms more stable micelles than P-PVP (at similar MW, CMC values for S-PVP are lower than for P-PVP). It was found that amphiphilic PVP incorporated into negatively charged liposomes effectively prevents polycation(poly-ethylpyridinium-4-vinylchloride)-induced liposome aggregation, completely abolishing it at ca. 10 mol% polymer content in liposomes. Additionally, the liposome-incorporated PVP prevents the fluorescence quenching of the membrane-incorporated hydrophobic fluorescent label [N-(4-fluoresceinthiocarbamoyl)dipalmitoyl-PE] by the free polycation. PVP-modified liposomes were loaded with a self-quenching concentration of carboxyfluorescein, and their destabilization in the presence of mouse serum was investigated following the release of free dye. Amphiphilic PVP with MW between 1,500 and 8,000 provides good steric protection for liposomes. The degree of this protection depends on both polymer concentration and molecular size of the PVP block.     

A novel cholinesterase and brain-selective monoamine oxidase inhibitor for the treatment of dementia comorbid with depression and Parkinson's disease

Degeneration of cholinergic cortical neurons is one of the main reasons for the cognitive deficit in dementia of the Alzheimer type (AD) and in dementia with Lewy bodies (DLB). Many subjects with AD and DLB have extrapyramidal dysfunction and depression resulting from degeneration of dopaminergic, noradrenergic and serotoninergic neurons. We prepared a novel drug, TV-3326 (N-propargyl-3R-aminoindan-5yl)-ethyl methylcarbamate), with both cholinesterase (ChE) and monoamine oxidase (MAO) inhibitory activity, as potential treatment of AD and DLB. TV-3326 inhibits brain acetyl and butyrylcholinesterase (BuChE) in rats after oral doses of 10-100 mg/kg. After chronic but not acute treatment, it inhibits MAO-A and -B in the brain by more than 70% but has almost no effect on these enzymes in the small intestine in rats and rabbits. The brain selectivity results in minimal potentiation of the pressor response to oral tyramine. TV-3326 acts like other antidepressants in the forced swim test in rats, indicating a potential for antidepressant activity. Chronic treatment of mice with TV-3326 (26 mg/kg) prevents the destruction of nigrostriatal neurons by the neurotoxin MPTP (N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine). In addition to ChE and MAO inhibition, the propargylamine moiety of TV-3326 confers neuroprotective activity against cytotoxicity induced by ischemia and peroxynitrite in cultured neuronal cells that results from prevention of the fall in mitochondrial membrane potential and antiapoptotic activity. These unique multiple actions of TV-3326 make it a potentially useful drug for the treatment of dementia with Parkinsonian-like symptoms and depression.     

Unique resistance of I/LnJ mice to a retrovirus is due to sustained interferon gamma-dependent production of virus-neutralizing antibodies

Selection of immune escape variants impairs the ability of the immune system to sustain an efficient antiviral response and to control retroviral infections. Like other retroviruses, mouse mammary tumor virus (MMTV) is not efficiently eliminated by the immune system of susceptible mice. In contrast, MMTV-infected I/LnJ mice are capable of producing IgG2a virus-neutralizing antibodies, sustain this response throughout their life, and secrete antibody-coated virions into the milk, thereby preventing infection of their progeny. Antibodies were produced in response to several MMTV variants and were cross-reactive to them. Resistance to MMTV infection was recessive and was dependent on interferon (IFN)-gamma production, because I/LnJ mice with targeted deletion of the INF-gamma gene failed to produce any virus-neutralizing antibodies. These findings reveal a novel mechanism of resistance to retroviral infection that is based on a robust and sustained IFN-gamma-dependent humoral immune response.     

Chain-Breaking Antioxidant Capability of Some Beverages as Determined by the Clark Electrode Technique

The protective effects of red wine, tea, and coffee on cancer, atheroclerosis, and other diseases are attributed to the antioxidant activity (AOA) of polyphenols, which are abundant in those beverages. We present a rational procedure for determining the total chain-breaking AOA of beverages with the use of the Clark electrode technique. The procedure is based on the steady monitoring of oxygen consumption accompanying the chain peroxidation of methyl linoleate in Triton X-100 aqueous micelles induced by 2,2'-azobis(2-amidinopropan) dihydrochloride as a source of active free radicals. AOA was characterized by the number of kinetic chains (expressed in concentration units) that could be terminated by a beverage. The procedure was applied to determine the AOA of nine red wines and single samples of green and black teas, white wine, beer, and soluble coffee. The addition of any of the studied beverages to the testing system resulted in pronounced retardation of methyl linoleate oxidation. The period of increase in the oxidation rate with time caused by antioxidant consumption was preceded by a period when the oxidation rate was visibly decreased with time (increase in inhibition). The release of polyphenols from their complexes with other components of beverages was suggested as the most probable mechanism of this event, which was observed for the first time in this study.     

Phylogeographical structure of the tick Ixodes persulcatus: A novel view

The tick Ixodes persulcatus Schulze, 1930, has a wide distribution from the Baltic to the Far East and is a vector of a number of human pathogens. Thus, the study of the genetic structure and evolution of this species is of great epidemiological importance. rRNA genes were used as genetic markers to identify the phylogeographical structure of the ticks. The sequences of gene fragments of 28S (expansion segment D3) and mitochondrial 12S rRNA for 25 and 76 ticks, respectively, that had been collected in various regions of Russia in 2007–2011, were obtained. The sequences of the 28S rRNA D3 segment were identical for all ticks within the studied area. Analysis of the sequences of the mitochondrial 12S rRNA fragment revealed 4 haplotypes with one occurring at a frequency of 0.96. It is shown that the ‘deep’ population structure of I. persulcatus (McLain et al., 2001) was erroneous because of the inclusion of contaminating fungi sequences of 28S rRNA in the phylogenetic analysis. This was, possibly, due to the use of universal PCR primers that amplify the DNA of a wide range of eukaryotes, particularly of fungi which are common in samples of ticks. The influence of PCR conditions on the preferential amplification of the DNA of different organisms is also demonstrated.