Duplexes of 21 nucleotide RNA specific for COX II mediates RNA interference in cultured bovine aortic coronary endothelial cells (BAECs)

The present study was conducted to utilize a double-stranded RNA (dsRNA) specific for cyclooxygenase (COX) II and demonstrate inhibition of the expression of COX II protein and its product PGE. A 21-dsRNA specific for COX II was introduced by lipofectamine into a primary cell culture of bovine aortic coronary endothelial cells (BAECs). BAECs basally express COX I but not COX II, and COX II expression is only apparent after stimulation with phorbol 12-myristate acetate (PMA). We first demonstrated that the lipofected fluorescent dsRNA-COX II is accumulated and localized within the cultured cells. We then demonstrated gene silencing of PMA-induced COX II protein expression by dsRNA-COX II using immuno-histochemistry. Western blot analysis and radioimmunoassay were used to quantitate the percent of inhibition. It was found that lipofected dsRNA-COX II reduced the percent of PMA-induced COX II enzyme by 36% and PGE production by 40%. There was no demonstrable effect of dsRNA-COX II or PMA on COX I expression.     

Novel methodology for the follow-up of acute lymphoblastic leukemia using FTIR microspectroscopy

In this report, we present a novel spectroscopic method of follow-up during chemotherapy treatment for B- and T-cell childhood leukemia patients. We isolated peripheral lymphocytes from blood drawn from patients before and after the chemotherapy and collected Microscopic FTIR (FTIR-MC) spectra of the isolated lymphocytes. Our results showed that nucleic acids content decreased in both types of patients. Changes in phospholipids and proteins level could be observed. The overall effects of drugs administered to the patients can be understood at the molecular level using FTIR-MC and these results are expected to stimulate wider applications of spectroscopy in leukemia research.     

Combined use of micro-preparative gel electrophoresis and reversed-phase high-performance liquid chromatography for purification of amyloid beta peptides deposited in brains of Alzheimer's disease patients

A new micro-technique is developed for purification of amyloid beta peptides (A beta) extracted from brain tissues of patients with Alzheimer's disease (AD). It includes SDS-polyacrylamide gel electrophoresis of the extracted brain tissue material, electroblotting onto supporting membranes, and reversed-phase HPLC of the proteins eluted from membranes. By this technique, the extracted A beta are first separated electrophoretically from the higher and lower molecular mass tissue components, and then purified by reversed-phase HPLC from the contaminants having similar molecular masses, but different retention times on the column. In contrast to the common large-scale isolation procedures employing density gradient centrifugation, enzymatic digestions and size-exclusion chromatography, the developed micro-technique might be applied for biochemical analysis of A beta contained in small AD brain tissue specimens.     

An optimized model for hCG stimulation of human mural granulosa cell culture

Ovarian follicular development and ovulation in mammals is a highly-regulated process. Most of the current knowledge of ovarian processes was obtained from the studies of non-human models. Molecular studies on human ovarian processes suffer from lack of material and appropriate research tools. Mural granulosa cells (MGCs) culture is a major tool for studying the effect of different substances but a major problem for using these primary MGCs is their unresponsiveness to hCG stimulation at the time of oocyte retrieval. It is acceptable that MGCs regain responsiveness during days in culture but when the best time is and how to accelerate the regenerative process are unknown.The aim of the current study was to establish an optimized protocol which will provide a practical and efficient tool to examine the effect of LH/hCG on different downstream targets in luteinized MGCs.hCG effects were examined according to days in culture and hCG stimulation time. As read-out, we analyzed the gene expression of known hCG targets, protein production, and progesterone secretion.Our results show that with a daily medium exchange, the strongest effect was achieved already 4 days after seeding. On day 4, hCG stimulation triggers two major patterns of gene expression. Early induced genes were highly expressed 6–8?h after hCG, while 24?h of hCG stimulation was needed for late induced genes.Based on our results, we suggest daily medium exchange for 4 days before adding hCG and examine its effect 6 and 24?h later.     

A High-Resolution Genetic Map of the Familial Mediterranean Fever Candidate Region Allows Identification of Haplotype-Sharing among Ethnic Groups

Familial Mediterranean fever (FMF) is a recessive disorder of inflammation caused by mutations in a gene (designatedMEFV) on chromosome 16p13.3. We have recently constructed a 1-Mb cosmid contig that includes the FMF critical region. Here we show genotype data for 12 markers from our physical map, including 5 newly identified microsatellites, in FMF families. Intrafamilial recombinations placedMEFVin the ∼285 kb betweenD16S468/D16S3070andD16S3376.We observed significant linkage disequilibrium in the North African Jewish population, and historical recombinants in the founder haplotype placedMEFVbetweenD16S3082andD16S3373(∼200 kb). In smaller panels of Iraqi Jewish, Arab, and Armenian families, there were significant allelic associations only forD16S3370andD16S2617among the Armenians. A sizable minority of Iraqi Jewish and Armenian carrier chromosomes appeared to be derived from the North African Jewish ancestral haplotype. We observed a unique FMF haplotype common to Iraqi Jews, Arabs, and Armenians and two other haplotypes restricted to either the Iraqi Jewish or the Armenian population. These data support the view that a few major mutations account for a large percentage of the cases of FMF and suggest that some of these mutations arose before the affected Middle Eastern populations diverged from one another.     

Influence of Cell Polarity on Retrovirus-Mediated Gene Transfer to Differentiated Human Airway Epithelia

Gene transfer with recombinant murine leukemia viruses (MuLV) provides the potential to permanently correct inherited lung diseases, such as cystic fibrosis (CF). Several problems prevent the application of MuLV-based recombinant retroviruses to lung gene therapy: (i) the lack of cell proliferation in mature pulmonary epithelia, (ii) inefficient gene transfer with a vector applied to the apical surface, and (iii) low titers of many retroviral preparations. We found that keratinocyte growth factor (KGF) stimulated proliferation of differentiated human tracheal and bronchial epithelia. Approximately 50% of epithelia divided in response to KGF as assessed by bromodeoxyuridine histochemistry. In airway epithelia stimulated to divide with KGF, high-titer ampho- and xenotropic enveloped vectors preferentially infected cells from the basal side. However, treatment with hypotonic shock or EGTA transiently increased transepithelial permeability, enhancing gene transfer with the vector applied to the mucosal surfaces of KGF-stimulated epithelia. Up to 35% of cells expressed the transgene after gene transfer. By using this approach, cells throughout the epithelial sheet, including basal cells, were targeted. Moreover, the Cl⁻ transport defect in differentiated CF airway epithelia was corrected. These findings suggest that barriers to apical infection with MuLV can be overcome.