NF-kappaB-mediated IAP expression induces resistance of intestinal epithelial cells to apoptosis after polyamine depletion

Apoptosis plays a crucial role in maintenance of intestinal epithelial integrity and is highly regulated by numerous factors, including cellular polyamines. We recently showed that polyamines regulate nuclear factor (NF)-kappaB activity in normal intestinal epithelial (IEC-6) cells and that polyamine depletion activates NF-kappaB and promotes resistance to apoptosis. The current study went further to determine whether the inhibitors of apoptosis (IAP) family of proteins, c-IAP2 and XIAP, are downstream targets of activated NF-kappaB and play a role in antiapoptotic activity of polyamine depletion in IEC-6 cells. Depletion of cellular polyamines by alpha-difluoromethylornithine not only activated NF-kappaB activity but also increased expression of c-IAP2 and XIAP. Specific inhibition of NF-kappaB by the recombinant adenoviral vector containing IkappaBalpha superrepressor (AdIkappaBSR) prevented the induction of c-IAP2 and XIAP in polyamine-deficient cells. Decreased levels of c-IAP2 and XIAP proteins by inactivation of NF-kappaB through AdIkappaBSR infection or treatment with the specific inhibitor Smac also overcame the resistance of polyamine-depleted cells to apoptosis induced by the combination of tumor necrosis factor (TNF)-alpha and cycloheximide (CHX). Although polyamine depletion did not alter levels of procaspase-3 protein, it inhibited formation of the active caspase-3. Decreased levels of c-IAP2 and XIAP by Smac prevented the inhibitory effect of polyamine depletion on the cleavage of procaspase-3 to the active caspase-3. These results indicate that polyamine depletion increases expression of c-IAP2 and XIAP by activating NF-kappaB in intestinal epithelial cells. Increased c-IAP2 and XIAP after polyamine depletion induce the resistance to TNF-alpha/CHX-induced apoptosis, at least partially, through inhibition of the caspase-3 activity.     

Ventral approach to the lumbar spine of the Sprague-Dawley rat

Lumbar intervertebral disc repair is an important tissue-engineering research area. In creating an in vivo rat model to evaluate repair techniques, the authors developed a surgical transperitoneal approach that permits the easy exposure of four lumbar vertebral bodies with no surgery-related peri- or postoperative complications.     

The myostatin gene is a downstream target gene of basic helix-loop-helix transcription factor MyoD

Myostatin is a negative regulator of myogenesis, and inactivation of myostatin leads to heavy muscle growth. Here we have cloned and characterized the bovine myostatin gene promoter. Alignment of the upstream sequences shows that the myostatin promoter is highly conserved during evolution. Sequence analysis of 1.6 kb of the bovine myostatin gene upstream region revealed that it contains 10 E-box motifs (E1 to E10), arranged in three clusters, and a single MEF2 site. Deletion and mutation analysis of the myostatin gene promoter showed that out of three important E boxes (E3, E4, and E6) of the proximal cluster, E6 plays a significant role in the regulation of a reporter gene in C(2)C(12) cells. We also demonstrate by band shift and chromatin immunoprecipitation assay that the E6 E-box motif binds to MyoD in vitro and in vivo. Furthermore, cotransfection experiments indicate that among the myogenic regulatory factors, MyoD preferentially up-regulates myostatin promoter activity. Since MyoD expression varies during the myoblast cell cycle, we analyzed the myostatin promoter activity in synchronized myoblasts and quiescent "reserve" cells. Our results suggest that myostatin promoter activity is relatively higher during the G(1) phase of the cell cycle, when MyoD expression levels are maximal. However, in the reserve cells, which lack MyoD expression, a significant reduction in the myostatin promoter activity is observed. Taken together, these results suggest that the myostatin gene is a downstream target gene of MyoD. Since the myostatin gene is implicated in controlling G(1)-to-S progression of myoblasts, MyoD could be triggering myoblast withdrawal from the cell cycle by regulating myostatin gene expression.     

The role of RNA editing by ADARs in RNAi

Adenosine deaminases that act on RNA (ADARs) are RNA-editing enzymes that deaminate adenosines to create inosines in double-stranded RNA (dsRNA). Here we demonstrate that ADARs are not required for RNA interference (RNAi) and that they do not antagonize the pathway to a detectable level when RNAi is initiated by injecting dsRNA. We find, however, that transgenes expressed in the somatic tissues of wild-type animals are silenced in strains with deletions in the two genes encoding ADARs, adr-1 and adr-2. Transgene-induced gene silencing in adr-1;adr-2 mutants depends on genes required for RNAi, suggesting that a dsRNA intermediate is involved. In wild-type animals we detect edited dsRNA corresponding to transgenes, and we propose that editing of this dsRNA prevents somatic transgenes from initiating RNAi in wild-type animals.     

The axial injury tolerance of the human foot/ankle complex and the effect of Achilles tension

Axial loading of the foot/ankle complex is an important injury mechanism in vehicular trauma that is responsible for severe injuries such as calcaneal and tibial pilon fractures. Axial loading may be applied to the leg externally, by the toepan and/or pedals, as well as internally, by active muscle tension applied through the Achilles tendon during pre-impact bracing. The objectives of this study were to investigate the effect of Achilles tension on fracture mode and to empirically model the axial loading tolerance of the foot/ankle complex. Blunt axial impact tests were performed on forty-three (43) isolated lower extremities with and without experimentally simulated Achilles tension. The primary fracture mode was calcaneal fracture in both groups. However, fracture initiated at the distal tibia more frequently with the addition of Achilles tension (p < 0.05). Acoustic sensors mounted to the bone demonstrated that fracture initiated at the time of peak local axial force. A survival analysis was performed on the injury data set using a Weibull regression model with specimen age, gender, body mass, and peak Achilles tension as predictor variables (R2 = 0.90). A closed-form survivor function was developed to predict the risk of fracture to the foot/ankle complex in terms of axial tibial force. The axial tibial force associated with a 50% risk of injury ranged from 3.7 kN for a 65 year-old 5th percentile female to 8.3 kN for a 45 year-old 50th percentile male, assuming no Achilles tension. The survivor function presented here may be used to estimate the risk of foot/ankle fracture that a blunt axial impact would pose to a human based on the peak tibial axial force measured by an anthropomorphic test device.     

Horizontal transfer of mercury resistance genes in natural bacterial populations

The results of studying the horizontal transfer of mercury resistance determinants in environmental bacterial populations are reviewed. Identical or highly homologous mercury resistance (mer) operons and transposons were found in bacteria of different taxonomic groups from geographically distant regions. Recombinant mer operons and transposons were revealed. The data suggest high frequencies of horizontal transfer and of recombination for mercury resistance determinants. The mechanisms of horizontal gene transfer were elucidated in Gram-negative and Gram-positive bacteria. New transposons were found and analyzed.     

The influence of fluid shear on the structure and material properties of sulphate-reducing bacterial biofilms

Biofilms of sulphate-reducing Desulfovibrio sp. EX265 were grown in square section glass capillary flow cells under a range of fluid flow velocities from 0.01 to 0.4 m/s (wall shear stress, τ_w, from 0.027 to 1.0 N/m²). In situ image analysis and confocal scanning laser microscopy revealed biofilm characteristics similar to those reported for aerobic biofilms. Biofilms in both flow cells were patchy and consisted of cell clusters separated by voids. Length-to-width ratio measurements (l _c:w _c) of biofilm clusters demonstrated the formation of more “streamlined” biofilm clusters (l _c:w _c=3.03) at high-flow velocity (Reynolds number, Re, 1200), whereas at low-flow velocity (Re 120), the l _c:w _c of the clusters was approximately 1 (l _c:w _c of 1 indicates no elongation in the flow direction). Cell clusters grown under high flow were more rigid and had a higher yield point (the point at which the biofilm began to flow like a fluid) than those established at low flow and some biofilm cell aggregates were able to relocate within a cluster, by travelling in the direction of flow, before attaching more firmly downstream. Received 01 February 2002/ Accepted in revised form 16 July 2002     

Absolute and effective cross-sections for low-energy electron-scattering processes within condensed matter

Within the last two decades, a number of experimental techniques have been developed to measure mean free paths and absolute and effective cross-sections for various processes related to the interaction of low-energy electrons with condensed matter. In all of the experiments, a monochromatic electron beam impinges on a thin multilayer film composed of atoms and/or molecules condensed on a metal or semiconductor substrate held at cryogenic temperatures in an ultra-high-vacuum system. Depending on the apparatus, cross-sections are obtained from low-energy electron transmission (LEET), high-resolution electron energy loss (HREEL), x-ray photoelectron (XPS) spectroscopy, electron-stimulated desorption (ESD) of neutral and ions, or a combination of these techniques. Quasi-elastic and inelastic mean free paths have been extracted from LEET data. This method has also served to generate absolute cross-sections for electron trapping and fragment production from the dissociation of transient molecular anions. In amorphous ice, a complete set of absolute cross-sections for all inelastic losses by 1–20 eV electrons has been obtained from HREEL data. Effective cross-sections for neutral and ionic radical formation were generated by desorption and XPS experiments. These various methods are briefly described in this article, and the corresponding cross-sections in the range 0–20 eV summarized. Received: 10 September 1998 / Accepted: 22 October 1998     

The primary structure of fatty-acid-binding protein from nurse shark liver. Structural and evolutionary relationship to the mammalian fatty-acid-binding protein family.

The primary structure of a fatty-acid-binding protein (FABP) isolated from the liver of the nurse shark (Ginglymostoma cirratum) was determined by high-performance tandem mass spectrometry (employing multichannel array detection) and Edman degradation. Shark liver FABP consists of 132 amino acids with an acetylated N-terminal valine. The chemical molecular mass of the intact protein determined by electrospray ionization mass spectrometry (Mr = 15124 +/- 2.5) was in good agreement with that calculated from the amino acid sequence (Mr = 15121.3). The amino acid sequence of shark liver FABP displays significantly greater similarity to the FABP expressed in mammalian heart, peripheral nerve myelin and adipose tissue (61-53% sequence similarity) than to the FABP expressed in mammalian liver (22% similarity). Phylogenetic trees derived from the comparison of the shark liver FABP amino acid sequence with the members of the mammalian fatty-acid/retinoid-binding protein gene family indicate the initial divergence of an ancestral gene into two major subfamilies: one comprising the genes for mammalian liver FABP and gastrotropin, the other comprising the genes for mammalian cellular retinol-binding proteins I and II, cellular retinoic-acid-binding protein myelin P2 protein, adipocyte FABP, heart FABP and shark liver FABP, the latter having diverged from the ancestral gene that ultimately gave rise to the present day mammalian heart-FABP, adipocyte FABP and myelin P2 protein sequences. The sequence for intestinal FABP from the rat could be assigned to either subfamily, depending on the approach used for phylogenetic tree construction, but clearly diverged at a relatively early evolutionary time point. Indeed, sequences proximately ancestral or closely related to mammalian intestinal FABP, liver FABP, gastrotropin and the retinoid-binding group of proteins appear to have arisen prior to the divergence of shark liver FABP and should therefore also be present in elasmobranchs. The presence in shark liver of an FABP which differs substantially in primary structure from mammalian liver FABP, while being closely related to the FABP expressed in mammalian heart muscle, peripheral nerve myelin and adipocytes, opens a further dimension regarding the question of the existence of structure-dependent and tissue-specific specialization of FABP function in lipid metabolism.