Regulation of bovine brain microvascular endothelial tight junction assembly and barrier function by laminar shear stress

Blood-brain barrier (BBB) controls paracellular solute diffusion into the brain microenvironment and is maintained primarily by tight junctions between adjacent microvascular endothelial cells. Studies implicate blood flow-associated shear stress as a pathophysiological mediator of BBB function, although detailed biochemical data are scarce. We hypothesize that shear stress upregulates BBB function via direct modulation of expression and properties of pivotal tight-junction proteins occludin and zonula occludens-1 (ZO-1). Bovine brain microvascular endothelial cells (BBMvECs) were exposed to either steady or pulsatile shear stress (10 and 14 dyn/cm(2), respectively) for 24 h. Sheared BBMvECs were monitored for occludin-ZO-1 expression, association, and subcellular localization, and transendothelial permeability of BBMvECs to FITC-dextran and (14)[C]sucrose was assessed. Actin reorganization and BBMvEC realignment were observed following steady shear stress for 24 h. Substantial increases in occludin mRNA and protein expression (2.73 +/- 0.26- and 1.83 +/- 0.03-fold) and in occludin-ZO-1 association (2.12 +/- 0.15-fold) were also observed. Steady shear stress also induced clear relocalization of both proteins to the cell-cell border in parallel with reduced transendothelial permeability to FITC-dextran (but not sucrose). Following pulsatile shear stress, increased protein levels for both occludin and ZO-1 (2.15 +/- 0.02- and 1.67 +/- 0.21-fold) and increased occludin-ZO-1 association (2.91 +/- 0.14-fold) were observed in parallel with a reduction in transendothelial permeability to (14)[C]sucrose. Shear stress upregulates BBMvEC barrier function at the molecular level via modulation of expression, association, and localization of occludin and ZO-1. The pulsatile shear model appeared to give the most profound biochemical responses.     

Covalent cross-linking of AcVal-tRNA to Tetrahymena thermophila cytoplasmic ribosomes and two of its 17S rRNA mutants

Tetrahymena thermophila 80S ribosomes have been cross-linked to non-enzymatically bound AcVal-tRNA, presumably at the ribosomal P-site. Like the ribosomes from Escherichia coli, yeast, and Artemia salina, cross-linking is exclusively to C-1609, the equivalent of the E. coli C-1400 residue. Mutation of the RNA from G-1707 to A or from U-1711 to C which results in resistance to paromomycin or hygromycin, respectively, failed to affect the rate, yield, or site of cross-linking. The presence of the antibiotics during cross-linking also was without effect. It is concluded that at these two positions the base changes made do not interfere with the tertiary structure of the decoding site.     

Epithelial permeability induced by neutrophil transmigration is potentiated by hypoxia: Role of intracellular cAMP

Mucosal tissues, such as the lung and intestine, are primary targets for ischemic damage. Under these conditions, neutrophil (polymorphonuclear leukocyte; PMN) infiltration into the protective epithelium has been implicated as a pathophysiologic mediator. Because PMN transepithelial migration results in increased paracellular permeability, and because our previous data revealed that epithelial hypoxia enhances PMN transmigration, we hypothesized that macromolecular permeability may be altered in epithelium exposed to hypoxia and reoxygenation (H/R) in the presence of PMNs. Human intestinal epithelia (T84) were grown on permeable supports, exposed to cellular hypoxia (pO₂ 20 torr) for 0–72 hr, and examined for increases in PMN-evoked permeability by using standard flux assays. Increasing epithelial hypoxia potentiated PMN-induced permeability of labeled paracellular tracers (size range 3–500 kD). Such increases were blocked by monoclonal antibody (mAb) to the PMN integrin CD11b (82 ± 1% decreased compared with control mAb) and were partially blocked by anti-CD47 mAb(51 ± 1%). Assessment of barrier recovery revealed that monolayers exposed to H/R were significantly diminished in their ability to reseal following PMN transmigration (recovery of 36 ± 6% in H/R vs. 94 ± 2% in normoxic controls). Because intracellular cyclic AMP (cAMP) has been demonstrated to regulate epithelial permeability, and because PMN-derived compound(s), (i.e., 5′-adenosine monophosphate; AMP) elevate epithelial cAMP, we examined the impact of hypoxia on epithelial cAMP responses. These experiments revealed that hypoxic epithelia were diminished in their ability to generate cAMP, and pharmacologic elevation (8-bromo-cAMP) of intracellular cAMP in hypoxic cells normalized both PMN-induced permeability changes and restoration of barrier function. These results support a role for PMN in increased intestinal permeability associated with reperfusion injury and imply a substantial role for cAMP signaling in maintenance of permeability during PMN transmigration. J. Cell. Physiol. 176:76–84, 1998. © 1998 Wiley-Liss, Inc.     

Molecular Analysis of Pseudomonas aeruginosa: Epidemiological Investigation of Mastitis Outbreaks in Irish Dairy Herds

Pseudomonas aeruginosa is a pathogen in both humans and animals. This bacterium, most often associated with respiratory infections in cystic fibrosis patients, was found to be the causative agent in bovine mastitis outbreaks among 11 Irish dairy herds. Epidemiological findings suggested that the infection was spread to all herds by teat wipes that had been contaminated with this organism. Two molecular-typing strategies were used in an attempt to determine the genomic relationship(s), if any, of the P. aeruginosa strains isolated from the various herds and to verify whether the same strain was responsible for each outbreak. Thirty-six isolates from the mastitis outbreaks were tested and compared to fourteen clinical isolates from Cork University Hospital. With one exception, all outbreak-linked strains produced identical patterns when ribotyped with ClaI and PvuII enzymes. Eight of the clinical isolates gave the same ClaI ribotype pattern as the mastitis-causing strains. However, PvuII proved more discriminatory, with only the outbreak isolates producing identical patterns. Similar results were obtained with RW3A-primed DNA amplification fingerprinting, with all outbreak isolates except one displaying the same fingerprint array. The clinical strains produced several fingerprint patterns, all of which were different from those of the mastitis-causing isolates. Fine-resolution DNA fingerprinting with a fluorescence-labelled RW3A primer also identified a number of low-molecular-weight polymorphisms that would have remained undetected by conventional methods. These data support the view that the same P. aeruginosa strain was responsible for the mastitis outbreaks in all 11 herds.     

Molecular analysis of Pseudomonas aeruginosa: epidemiological investigation of mastitis outbreaks in Irish dairy herds.

Pseudomonas aeruginosa is a pathogen in both humans and animals. This bacterium, most often associated with respiratory infections in cystic fibrosis patients, was found to be the causative agent in bovine mastitis outbreaks among 11 Irish dairy herds. Epidemiological findings suggested that the infection was spread to all herds by teat wipes that had been contaminated with this organism. Two molecular-typing strategies were used in an attempt to determine the genomic relationship(s), if any, of the P. aeruginosa strains isolated from the various herds and to verify whether the same strain was responsible for each outbreak. Thirty-six isolates from the mastitis outbreaks were tested and compared to fourteen clinical isolates from Cork University Hospital. With one exception, all outbreak-linked strains produced identical patterns when ribotyped with ClaI and PvuII enzymes. Eight of the clinical isolates gave the same ClaI ribotype pattern as the mastitis-causing strains. However, PvuII proved more discriminatory, with only the outbreak isolates producing identical patterns. Similar results were obtained with RW3A-primed DNA amplification fingerprinting, with all outbreak isolates except one displaying the same fingerprint array. The clinical strains produced several fingerprint patterns, all of which were different from those of the mastitis-causing isolates. Fine-resolution DNA fingerprinting with a fluorescence-labelled RW3A primer also identified a number of low-molecular-weight polymorphisms that would have remained undetected by conventional methods. These data support the view that the same P. aeruginosa strain was responsible for the mastitis outbreaks in all 11 herds.     

Gastropod evolutionary rates and phylogenetic relationships assessed using partial 28S rDNA and histone H3 sequences

Sequence data for two segments of 28S and Histone H3 from 36 gastropod taxa, a chiton, two bivalves and Nautilus are used to test recently published morphology‐based phylogenetic hypotheses of gastropod relationships. Statistical results suggest that the accuracy of the available hypotheses could be improved. The data support the monophyly of the Patellogastropoda (true limpets), Euthyneura and the ‘higher’ vetigastropods and the polyphyly of the ‘Cocculiniformia’. The division of the gastropods into two major clades (Eogastropoda and Orthogastropoda) as has been proposed on morphological grounds is not supported, and neither the Caenogastropoda nor Heterobranchia is well supported. Within the Euthyneura, opisthobranchs are paraphyletic with respect to the pulmonates. The hot vent taxon, Depressigyra, groups with the lower vetigastropod Pleurotomaria in some analyses. Much of the variability in the 28S rDNA segments lies in discrete areas of the sequence. Forone of the segments, corresponding to positions 691–942 of the mosquito Aedes albopictus 28S sequence, the variable regions represent known expansion regions (D4 and D5). For the other segment, corresponding to positions 2259–2538 of the A. albopictus sequence, the variable area, which is found in the patellogastropods, vetigastropods and Nautilus, represents an unreported expansion region. The data show marked variability in the rate of evolution in both segments of the 28S rDNA, whether or not the expansion regions are included. The variability is largely clade specific. Rates are high in the patellogastropods, vetigastropods, the lower heterobranch ‘Heterostropha’ (Cornirostra and Philippea), Depressigyra and the deep sea cocculinid limpet Coccopigya and substantially lower in other taxa. Rate variation in the histone H3 data is less extreme. The correlation between evolutionary rates in the two 28S rDNA segments is very high, andis also significant for the the pairing of each of the 28S rDNA segments with H3. The rate variability may be due to differential selection but no causative factor has been identified. The histone H3 data have high codon usage bias. For all amino acids encoded by multiple codons, at least some triplets occur at a frequency of less than a quarter of their expected usage. For all three‐, four‐and sixfold degenerate amino acids, the most abundant triplet occurs at least twice as frequently as expected. Despite the usage bias, there is a large amount of apparent homoplasy in synonymous alternatives at both the first and third codon positions.     

Recombinant monoclonal antibody yield in transgenic tobacco plants is affected by the wounding response via an ethylene dependent mechanism

Variability in recombinant IgG yield in transgenic tobacco plants has previously been observed in relation to leaf position, and is interpreted as a function of ageing and the senescence process, leading to increasing protein degradation. Here, similar findings are demonstrated in plants of different ages, expressing IgG but not IgG-HDEL, an antibody form that accumulates within the endoplasmic reticulum. Antibody yields declined following wounding in young transgenic plants expressing IgG but not in those expressing IgG-HDEL. However, in mature IgG plants, the opposite was demonstrated, with significant boosts in yield, while mature IgG-HDEL plants could not be boosted. The lack of response in IgG-HDEL plants suggests that the changes induced by wounding occur post-translationally, and the findings might be explained by wounding responses that differ in plants according to their developmental stages. Plant mechanisms involved in senescence and wounding overlap to a significant degree and compounds such as ethylene, jasmonic acid and salicylic acid are important for mediating downstream effects. Treatment of transgenic plants with ethylene also resulted in a decrease in recombinant IgG yield, which was consistent with the finding that wounded plants could induce lower IgG yields in neighbouring non-wounded plants. Treatment with 1-MCP, an ethylene antagonist, abrogated the IgG yield drop that resulted from wounding, but had no effect on the more gradual IgG yield loss associated with increasing plant age.     

Targeting tetramer-forming GABPβ isoforms impairs self-renewal of hematopoietic and leukemic stem cells

Hematopoietic stem cells (HSCs) and leukemic stem cells (LSCs) are both capable of self-renewal, with HSCs sustaining multiple blood lineage differentiation and LSCs indefinitely propagating leukemia. The GABP complex, consisting of DNA binding GABPα subunit and transactivation GABPβ subunit, critically regulates HSC multipotency and self-renewal via controlling an essential gene regulatory module. Two GABPβ isoforms, GABPβ1L and GABPβ2, contribute to assembly of GABPα(2)β(2) tetramer. We demonstrate that GABPβ1L/β2 deficiency specifically impairs HSC quiescence and survival, with little impact on cell cycle or apoptosis in differentiated blood cells. The HSC-specific effect is mechanistically ascribed to perturbed integrity of the GABP-controlled gene regulatory module in HSCs. Targeting GABPβ1L/β2 also impairs LSC self-renewal in p210(BCR-ABL)-induced chronic myelogenous leukemia (CML) and exhibits synergistic effects with tyrosine kinase inhibitor imatinib therapy in inhibiting CML propagation. These findings identify the tetramer-forming GABPβ isoforms as specific HSC regulators and potential therapeutic targets in treating LSC-based hematological malignancy.