Targeted disruption of LIG-1 gene results in psoriasiform epidermal hyperplasia

We have designed a chimeric promoter that can be stimulated by various pro-inflammatory mediators and so drive the expression of therapeutic genes under inflammatory conditions. The promoter has two parts, the [-247/+20] fragment of the human type IIA secreted phospholipase A2 gene promoter, which is stimulated by the pro-inflammatory cytokine interleukin-1beta (IL-1beta), and a double peroxisome proliferator-activated receptor response element that is activated by some eicosanoids and by non-steroidal anti-inflammatory drugs (NSAIDs). Transfection experiments using rabbit articular chondrocytes in primary culture showed that this chimeric promoter produced a low basal activity and was induced by NSAIDs, WY-14643, IL-1beta, and 15-deoxy Delta12,14 prostaglandin J2. The latter two compounds stimulated the promoter synergistically.     

Group V phospholipase A2 induces leukotriene biosynthesis in human neutrophils through the activation of group IVA phospholipase A2

We reported previously that exogenously added human group V phospholipase A(2) (hVPLA(2)) could elicit leukotriene B(4) (LTB(4)) biosynthesis in human neutrophils (Han, S. K., Kim, K. P., Koduri, R., Bittova, L., Munoz, N. M., Leff, A. R., Wilton, D. C., Gelb, M. H., and Cho, W. (1999) J. Biol. Chem. 274, 11881-11888). To determine the mechanism of the hVPLA(2)-induced LTB(4) biosynthesis in neutrophils, we thoroughly examined the effects of hVPLA(2) and their lipid products on the activity of group IVA cytosolic PLA(2) (cPLA(2)) and LTB(4) biosynthesis under different conditions. As low as 1 nm exogenous hVPLA(2) was able to induce the release of arachidonic acid (AA) and LTB(4). Typically, AA and LTB(4) were released in two phases, which were synchronized with a rise in intracellular calcium concentration ([Ca(2+)](i)) near the perinuclear region and cPLA(2) phosphorylation. A cellular PLA(2) assay showed that hVPLA(2) acted primarily on the outer plasma membrane, liberating fatty acids and lysophosphatidylcholine (lyso-PC), whereas cPLA(2) acted on the perinuclear membrane. Lyso-PC and polyunsaturated fatty acids including AA activated cPLA(2) and 5-lipoxygenase by increasing [Ca(2+)](i) and inducing cPLA(2) phosphorylation, which then led to LTB(4) biosynthesis. The delayed phase was triggered by the binding of secreted LTB(4) to the cell surface LTB(4) receptor, which resulted in a rise in [Ca(2+)](i) and cPLA(2) phosphorylation through the activation of mitogen-activated protein kinase, extracellular signal-regulated kinase 1/2. These results indicate that a main role of exogenous hVPLA(2) in neutrophil activation and LTB(4) biosynthesis is to activate cPLA(2) and 5-lipoxygenase primarily by liberating from the outer plasma membrane lyso-PC that induces [Ca(2+)](i) increase and cPLA(2) phosphorylation and that hVPLA(2)-induced LTB(4) production is augmented by the positive feedback activation of cPLA(2) by LTB(4).     

Affinity selection of DNA-binding proteins from yeast genomic DNA libraries by improved lambda phage display vector

Phage display is a useful means of identifying and selecting proteins of interest that bind specific targets. In order to examine the potential of phage display for the genome-wide screening of DNA-binding proteins, we constructed yeast genomic libraries using lambda foo-based vectors devised in this work. After affinity selection using GAL4 UAS(G) as a probe, phages expressing GAL4 were enriched approximately 5 x 10(5)-fold from the library. Approximately 90% of polypeptides encoded in correct translation reading frames by the selected phages were known or putative polynucleotide-binding proteins. This result clearly indicates that the modified lambda phage display vector in combination with our enrichment technique has great potential for the enrichment of DNA-binding proteins in a sequence-specific manner.     

The relative importance of the deep and superficial vascular systems for delay of the transverse rectus abdominis musculocutaneous flap as demonstrated in a rat model

The use of some form of delay maneuver for "high-risk" patients before transfer of the superior pedicled lower transverse rectus abdominis musculocutaneous (TRAM) flap for breast reconstruction has augmented the rate of success in both the experimental and clinical arenas. A common method of vascular delay has been the bilateral division of both the superficial inferior epigastric and deep inferior epigastric vessels. Whether all of these must be divided to adequately effect the delay is unknown. For that matter, the relative importance of the superficial versus the deep vascular systems is unclear. To investigate this uncertainty, a delay was attempted in 61 Sprague-Dawley rats by division of either the superficial inferior epigastric or deep cranial epigastric vessels (the latter is the homologue to the human deep inferior epigastric) in unilateral or bilateral fashion. Division of the contralateral superficial inferior epigastric vessel resulted in significantly greater TRAM flap survival than either ipsilateral or bilateral superficial inferior epigastric vessel division (p = 0.0034 or p = 0.0093, respectively). Division of the ipsilateral or bilateral deep cranial epigastric vessel resulted in significantly greater flap survival than just contralateral deep cranial epigastric vessel division (p = 0.0034 or p = 0.006, respectively). No significant difference was observed between the group having contralateral superficial inferior epigastric or groups with ipsilateral deep cranial epigastric division, implying that either alone would be efficacious to achieve the desired delay effect. This would allow the other vascular system to be retained intact for later potential salvage maneuvers as needed.     

Dissociation of DNA damage and mitochondrial injury caused by hydrogen peroxide in SV-40 transformed lung epithelial cells

Background  

Since lung epithelial cells are constantly being exposed to reactive oxygen intermediates (ROIs), the alveolar surface is a major site of oxidative stress, and each cell type may respond differently to oxidative stress. We compared the extent of oxidative DNA damage with that of mitochondrial injury in lung epithelial cells at the single cell level.     

Mechanical vibration model for chromosomes in metaphase of mitosis and possible application to the interruption of cell division

The behavior of chromosomes subjected to external mechanical vibration in metaphase of mitosis is modeled, and its possible applications to the interruption of cell divisions is investigated. The modeling is based on a mass and string coupled system where the mass and the string correspond to the condensed chromosome and the kinetochore microtubule, respectively. After establishing the motion of equation for a chromosome, the frequency response to the forced vibration from the outside of the cell is formulated. This will lead to an efficient method for kinetic energy transfer to chromosomes by finding the resonant frequencies of the cell system. There is a possibility that the increased kinetic energy in one chromosome will affect the cell as a whole, thereby interrupting the mitosis by the mechanisms that are discussed in this report. Rough calculation shows that the vibration in the frequency range of 22-50 kHz is effective for controlling the mitosis of human cells.     

Cholesterol,oxidative stress,and Alzheimer's disease: expanding the horizons of pathogenesis

Recent epidemiological, clinical, and experimental data suggest that cholesterol may play a role in Alzheimer's disease (AD). We have recently shown that cholesterolemia has a profound effect in the development and modulation of amyloid pathology in a transgenic model of AD. This review summarizes recent advancements in our understanding of the potential role of cholesterol and the amyloid beta protein in initiating the generation of free radicals and points out their role in a chain of events that causes damage of essential macromolecules in the central nervous system and culminates in neuronal dysfunction and loss. Experimental data links cholesterol and oxidative stress with some neurodegenerative aspects of AD.