Neurons are protected from excitotoxic death by p53 antisense oligonucleotides delivered in anionic liposomes.

The potential of anionic liposomes for oligonucleotide delivery was explored because the requirement for a net-positive charge on transfection-competent cationic liposome-DNA complexes is ambiguous. Liposomes composed of phosphatidylglycerol and phosphatidylcholine were monodisperse and encapsulated oligonucleotides with 40-60% efficiency. Ionic strength, bilayer charge density, and oligonucleotide chemistry influenced encapsulation. To demonstrate the biological efficacy of this vector, antisense oligonucleotides to p53 delivered in anionic liposomes were tested in an in vitro model of excitotoxicity. Exposure of hippocampal neurons to glutamate increased p53 protein expression 4-fold and decreased neuronal survival to approximately 35%. Treatment with 1 microm p53 antisense oligonucleotides in anionic liposomes prevented glutamate-induced up-regulation of p53 and increased neuronal survival to approximately 75%. Encapsulated phosphorothioate p53 antisense oligonucleotides were neuroprotective at 5-10-fold lower concentrations than when unencapsulated. Replacing the anionic lipid with phosphatidylserine significantly decreased neuroprotection. p53 antisense oligonucleotides complexed with cationic liposomes were ineffective. Neuroprotection by p53 antisense oligonucleotides in anionic liposomes was comparable with that by glutamate receptor antagonists and a chemical inhibitor of p53. Anionic liposomes were also capable of delivering plasmids and inducing transgene expression in neurons. Anionic liposome-mediated internalization of Cy3-labeled oligonucleotides by neurons and several other cell lines demonstrated the universal applicability of this vector.     

Antisense raf oligodeoxyribonucleotide is a radiosensitizer in vivo.

Raf-1, a cytosolic protein serine/threonine kinase, plays important roles in cell growth, proliferation, transformation, and cell survival. The aim of the present study was to evaluate the radiotherapeutic efficacy of a fully phosphorothioated and well-characterized antisense raf oligodeoxyribonucleotide (ODN) corresponding to the 3'-untranslated region of human c-raf-1 mRNA (ISIS 5132/5132). Using our recently developed liposome encapsulation of ODN approach, we first compared the pharmacokinetic parameters of a liposomal formulation of 5132 (LE-5132) and 5132. The peak plasma concentrations 5 minutes after ODN administrations (30 mg/kg i.v.) were 28.5 microg/ml and 13.5 microg/ml for LE-5132 and 5132, respectively. The decrease in plasma concentration of LE-5132 and 5132 followed a biexponential pattern, with initial distribution half-lives (t1/2alpha) of 34.8 minutes and 21.6 minutes, respectively. The terminal half-lives (t1/2beta) with LE-5132 and 5132 were 14.5 hours and 4.3 hours, respectively. The area under the plasma concentration-time curve (AUC) was 5.8 times higher with LE-5132 than with 5132. Significantly higher intact ODN levels could be measured in most organs within 48 hours of administration of LE-5132 compared with 5132 (liver 18.4-fold, spleen, 31-fold, heart 3-fold, lungs 1.5-fold). In kidneys, the level was lower with LE-5132 (0.77-fold). LE-5132 composition, unlike 5132, did not affect clotting time in vitro. Significant decline in the level of Raf-1 protein was observed in vitro in relatively radioresistant human laryngeal squamous cell carcinoma cells (SQ-20B) treated with LE-5132 compared with SQ-20B cells treated with equimolar concentration of 5132 or liposome-encapsulated mismatched 5132 (0.5 microM LE-5132, 71.3%+/-22.5%; 1.0 microM LE-5132, 79.6%+/-16.7%). In addition, LE-5132 appeared to be a more potent antitumor compound than 5132 (p < 0.001). These data established the suitability of LE-5132 for in vivo radiotherapeutic efficacy studies. Intravenous administration of LE-5132 into SQ-20B tumor-bearing athymic mice inhibited Raf-1 expression in tumor tissue compared with blank liposome-treated or untreated control groups. LE-5132 or ionizing radiation (IR) treatment alone caused significant but transient inhibition of SQ-20B tumor growth but not tumor regression. Remarkably, a combination of LE-5132 and IR treatments led to significant and sustained tumor regression for at least 27 days after the last treatment (< 0.001). Histopathologic examination of tumor samples revealed a significant proportion of cells containing fragmented chromatin in the LE-5132 + IR treatment group as compared with single agent and untreated control groups. These in vivo data support the notion that Raf-1 has proliferative and survival functions and advance the scientific and technologic bases for the use of antisense raf ODN in the management of radioresistant malignancies.     

Effects of environmental and spatial gradients on Quercus-dominated Mountain forest communities in the Hindu-Kush ranges of Pakistan

Quercus-dominated forests are among the most important broad-leaved evergreen forests of the Hindu Kush ranges and are currently prone to drastic anthropogenic and climatic changes. The aim of this study was to provide basic data for the development of a regional oak forest ecosystem framework for ecological restoration and management plan development to maintain local peoples’ livelihoods. Hence, we analyzed distribution patterns and environmental factors that affect regional oak forests’ species composition and diversity. Ward’s Agglomerative clustering divided oak-dominated forest communities into three groups: i.e., Group I, dominated by Quercus baloot had an importance value index (IVI) of 89.87 ± 4.31, Group II, dominated by Quercus dilatata had an IVI of 32.16 ± 15.01, and Group III, dominated by Quercus oblongata had an IVI of 83.14 ± 4.67, respectively. The environmental factors which vary significantly within these communities were latitude, elevation, clay content and bulk density of the soil. Wilting point, saturation point, and electrical conductivity were also considered as ecosystem structural variables. Canonical correspondence analysis (CCA) indicated that community structure was affected by various environmental factors including precipitation, slope angle, elevation, clay content, and relative humidity.     

Demonstration of aromatic l-amino acid decarboxylase activity in human brain with l-dopa and l-5-hydroxytryptophan as substrates by high-performance liquid chromatography with electrochemical detection

The enzymatic decarboxylations of l-DOPA and l-5-hydroxytryptophan (l-5-HTP) by aromatic l-amino acid decarboxylase (AADC) were measured with homogenates from human brain regions, caduate nucleus and hypothalamus, using our new and highly sensitive methods for l-DOPA decarboxylase and l-5-HTP decarboxylase by high-performance liquid chromatography with electrochemical detection (HPLC-ED). Dopamine formed from l-DOPA as substrate was measured for DOPA decarboxylase activity using d-DOPA for the blank. For 5-HTP decarboxylase activity, serotonin (5-HT) formed from l-5-HTP was measured, and the blank value in presence of NSD-1055 was subtracted. NSD-1055 inhibited 5-HTP decarboxylase activity completely at a concentration of 0.2 mM. In this study, the properties of l-5-HTP decarboxylase activity in human caudate nucleus were first examined. AADC activities in human brains were found to be widely variable for both l-DOPA and l-5-HTP as substrates. The ratio of the activities for l-DOPA and l-5-HTP were found to be significantly higher in hypothalamus than in caudate nucleus. AADC activity for l-DOPA in the brain was found to be linear up to 40 min of incubation, while that for l-5-HTP was found to be linear up to 240 min of incubation. The optimum pyridoxal phosphate concentration was found to be similar for both substrates and was between 0.01 and 0.1 mM. The optimum pH values were found to be 7.2 and 8.2 for l-DOPA decarboxylase and l-5-HTP decarboxylase, respectively. K_m and V_max values for a human caudate nucleus l-DOPA decarboxylase were found to be 414 μM and 482 pmol/min/g wet weight, respectively, while those for l-5-HTP decarboxylase were found to be 90 μM and 71 pmol/min/g wet weight, respectively.     

Deletion and translocation of chromosome 11q13 sequences in cervical carcinoma cell lines.

Molecular genetic studies on HeLa cell-derived nontumorigenic and tumorigenic hybrids have previously localized the HeLa cell tumor-suppressor gene to the long arm of chromosome 11. Extensive molecular and cytogenetic studies on HeLa cells have shown chromosome band 11q13 to be rearranged in this cell line. To determine whether q13 rearrangement is a nonrandom event in cervical carcinomas, six different human papilloma virus (HPV)-positive (HeLa, SiHa, Caski, C4-I, Me180, and Ms751) and two different HPV-negative (C33A and HT3) cell lines were studied. Long-range restriction mapping using a number of q13-specific probes showed molecular rearrangements within 75 kb of INT2 probe in three HPV-positive cell lines (HeLa, SiHa, and Caski) and in an HPV-negative cell line (HT3). FISH using an INT2 YAC identified a breakpoint within the sequences spanned by this YAC in two of the cell lines, HeLa and Caski. INT2 cosmid derived from this YAC showed deletion of cosmid sequences in two other cell lines, SiHa and C33A. These two cell lines, however, retained cosmid sequences of Cyclin D1, a probe localized 100 kb proximal to INT2. Deletions being the hallmark of a tumor-suppressor gene, we conclude that the 100-kb interval between the two cosmids might contain sequences of the cervical carcinoma tumor-suppressor gene.     

Improvement of Erythrose Reductase Activity,Deletion of By-products and Statistical Media Optimization for Enhanced Erythritol Production from Yarrowia lipolytica Mutant 49

The purpose of the present investigation was to produce erythritol by Yarrowia lipolytica mutant without any by-products. Mutants of Y. lipolytica were generated by ultra-violet for enhancing erythrose reductase (ER) activity and erythritol production. The mutants showing the highest ER activity were screened by triphenyl tetrazolium chloride agar plate assay. Productivity of samples was analyzed by thin-layer chromatography and high-performance liquid chromatography equipped with the refractive index detector. One of the mutants named as mutant 49 gave maximum erythritol production without any other by-products (particularly glycerol). Erythritol production and specific ER activity in mutant 49 increased to 1.65 and 1.47 times, respectively, in comparison with wild-type strain. The ER gene of wild and mutant strains was sequenced and analyzed. A general comparison of wild and mutant gene sequences showed the replacement of Asp²⁷⁰ with Glu²⁷⁰ in ER protein. In order to enhance erythritol production, we used a three component-three level-one response Box–Behnken of response surface methodology model. The optimum medium composition for erythritol production was found to be (g/l) glucose 279.49, ammonium sulfate 9.28, and pH 5.41 with 39.76 erythritol production.     

反相高效液相色谱法测定Bacillus claussi的DNA G+Cmol%

目的:为了建立细菌DNA G Cmol%的快速准确的测定方法,选用反相高效液相色谱法测定Bacillus claussiDNA的G C mol%。方法:以已完成全基因测序的Bacillus haloduransC-125作为标准菌株,作者实验室新分离鉴定的2株Bacillus claussi作为待测菌株。采用80 mmol/L磷酸二氢钾溶液(pH5.6)20%甲醇为移动相,检测波长260nm,流速1ml.min-1,在Kromasil C18柱上对4种碱基进行分离。结果:DNA碱基分离效果好,无杂峰干扰,以峰面积计算得到标准菌株Bacillus haloduransC-125的G C mol%为44.23%,待测菌株的G C mol%分别为45.75%、43.35%,经过统计学分析,与已经报道的G C mol%差异不显著。结论:实验结果充分显示高效反相液相色谱法测定细菌DNAG C mol%快速、准确、结果稳定,是细菌精确分类鉴定的一种可靠方法。