Use of cell wall stress to characterize σ22 (AlgT/U) activation by regulated proteolysis and its regulon in Pseudomonas aeruginosa

MucA sequesters extracytoplasmic function (ECF) σ²² ( algT/U encoded) from target promoters including P algD for alginate biosynthesis. We have shown that cell wall stress (e.g. d -cycloserine) is a potent inducer of the algD operon. Here we showed that MucB, encoded by the algT-mucABCD operon, interacts with MucA in the sigma–sequestration complex. We hypothesized that AlgW protease (a DegS homologue) is activated by cell wall stress to cleave MucA and release σ²². When strain PAO1 was exposed to d -cycloserine, MucA was degraded within just 10 min, and σ²² was activated. However, in an algW mutant, MucA was stable with no increased σ²² activity. Studies on a yaeL mutant, defective in an RseP/YaeL homologue, suggest that YaeL protease cleaves MucA only after cleavage by AlgW. A defect in mucD , encoding a periplasmic HtrA/DegP homologue, caused MucA instability, suggesting MucD degrades cell wall stress signals. Overall, these data indicate that cell wall stress signals release σ²² by regulated intramembrane proteolysis (RIP). Microarray analyses identified genes of the early and late cell wall stress stimulon, which included genes for alginate production. The subset of genes in the σ²² regulon was then determined, which included gene products predicted to contribute to recovery from cell wall stress.     

Dexamethasone Increases Adrenalectomy-Depressed Na+,K+-ATPase mRNA and Ouabain Binding in Spinal Cord Ventral Horn

Abstract: The Na⁺,K⁺-ATPase plays a key role in the regulation of ion fluxes and membrane repolarization in the CNS. We have studied glucocorticoid effects on biosynthesis of the Na⁺,K⁺-ATPase and on ouabain binding in the ventral horn of the spinal cord using intact rats, adrenalectomized (ADX) rats, and ADX rats receiving dexamethasone (ADX + DEX) during 4 days. Cryostat sections from spinal cords were incubated with a ³⁵S-oligonucleotide coding for the α₃-subunit or a ³H-cDNA coding for the β₁-subunit of the Na⁺,K⁺-ATPase using in situ hybridization techniques. In ventral horn motoneurons, grain density per cell and grain density per area of some for both probes were slightly reduced in ADX rats but significantly increased in the ADX + DEX group, using ANOVA and the Bonferroni's test. Statistical analysis of frequency histograms of neuronal densities further indicated a significant shift to the right for intact rats compared with ADX rats for both probes. Concomitantly, [³H]ouabain binding to membrane preparations from ventral horns was reduced in ADX rats and restored to normal by DEX administration. No effect of adrenalectomy or DEX treatment was obtained in the dorsal horn. In conclusion, glucocorticoids positively modulate the mRNA for the α₃-subunit and the β₁-subunit of the Na⁺,K⁺-ATPase and recover ouabain binding to normal values. The increments of the synthesis and activity of an enzyme affecting membrane repolarization and synaptic neurotransmission are consistent with the alleged stimulatory effect of glucocorticoids on spinal cord function.     

Multipotent mesenchymal stem cells from adult human eye conjunctiva stromal cells

Abstract Identification of mesenchymal stem cells (MSCs) derived from alternative sources has provided an exciting prospect for intensive investigation. This work focused on characterizing a new source of MSCs from stromal cells from human eye conjunctiva. In this study, after conjunctiva biopsies and culture of stromal segment of this tissue, fibroblast-like (SH2⁺, SH3⁺, CD29⁺, CD44⁺, CD166⁺, CD13⁺) human stromal cells, which can be differentiated toward the osteogenic, adipogenic, chondrogenic, and neurogenic lineages, were obtained. These cells expressed Oct-4, Nanog, Rex-1 genes, and some lineage-specific markers like cardiac actin and Keratin. Taken together, the results indicate that conjunctiva stromal-derived cells are a new source of multipotent MSCs and despite originating from an adult source, they express undifferentiated stem cell markers.     

Properties of the Sodium Extrusion Mechanism Controlled by Nerve Growth Factor in Chick Embryo Dorsal Root Ganglionic Cells

Abstract: Cell dissociates from embryonic chick dorsal root ganglia, incubated for 6 h with ²²Na⁺, accumulated four to six times more radioactivity in the absence than in the presence of Nerve Growth Factor (NGF). The accumulation of radioactivity paralleled the external Na⁺ concentration, indicating that the cells may have been reaching equilibrium with the medium. Delayed presentation of NGF to ²²Na⁺-loaded cells caused a rapid loss of radioactivity, even with extracellular ²²Na⁺ still present, demonstrating that NGF caused an overall efflux of Na⁺ rather than an accelerated equilibration. The Na⁺ exclusion from ²²Na⁺-loaded cells was dependent upon NGF concentration. Use of nutrient-rich medium, serum, and certain hormones and other proteins did not prevent the Na+ accumulation in the absence of NGF or its reversion by delayed NGF administration. Incubation of the ganglionic cells with ouabain or dinitrophenol during the ²²Na⁺ loading period (no NGF) increased the rate, but not the magnitude, of loading. The same incubation carried out in a Na+-free medium and followed by ²²Na⁺ presentation resulted in fast radioactive loading that was identical to that occurring in drug-free, NGF-deprived cells and was not prevented by presentation of NGF together with the ²²Na⁺. These data are consistent with a model in which NGF acts through a Na⁺ pump rather than by restricting Na+ influxes.     

Dominant ptsH mutations of the gene coding for synthesis of the HPr protein of the phosphoenolpyruvate-dependent phosphotransferase system in Escherichia coli K-12 merodiploids

Abstract The Escherichia coli ptsI and ptsH genes code for the synthesis of two proteins of the phosphoenolpyruvate-dependent phosphotransferase system (PTS), namely enzyme I and protein HPr. A number of ptsI ⁺ ptsH ⁺/F' ptsI ⁺ ptsH merodiploids was obtained. It was shown in experiments in vivo that ptsH mutations in the transposition are dominant. Bacterial extracts from these merodiploids supported [¹⁴C]methyl glucoside (MG) phosphorylation at the expense of phosphoenolpyruvate only half as much as extracts from the pts ⁺ cells. ptsI ⁺ ptsH /F' ptsI ⁺ ptsH ⁺ merodiploids appeared to be non-viable; the reason for this lack of viability is discussed.     

A role for chromosomal protein HMGN1 in corneal maturation

Abstract Corneal differentiation and maturation are associated with major changes in the expression levels of numerous genes, including those coding for the chromatin-binding high-mobility group (HMG) proteins. Here we report that HMGN1, a nucleosome-binding protein that alters the structure and activity of chromatin, affects the development of the corneal epithelium in mice. The corneal epithelium of Hmgn1 ^−/− mice is thin, has a reduced number of cells, is poorly stratified, is depleted of suprabasal wing cells, and its most superficial cell layer blisters. In mature Hmgn1 ^−/− mice, the basal cells retain the ovoid shape of immature cells, and rest directly on the basal membrane which is disorganized. Gene expression was modified in Hmgn1 ^−/− corneas: glutathione-S-transferase (GST)α 4and GST ω 1, epithelial layer-specific markers, were selectively reduced while E-cadherin and α-, β-, and γ-catenin, components of adherens junctions, were increased. Immunofluorescence analysis reveals a complete co-localization of HMGN1 and p63 in small clusters of basal corneal epithelial cells of wild-type mice, and an absence of p63 expressing cells in the central region of the Hmgn1 ^−/− cornea. We suggest that interaction of HMGN1 with chromatin modulates the fidelity of gene expression and affects corneal development and maturation.     

Analysis of splenic Gr-1int immature myeloid cells in tumor-bearing mice

It is known that the number of ImC, expressing myeloid markers, CD11b and Gr-1, increase with tumor growth and ImC play a role in the escape of tumor cells from immunosurveillance in tumor-bearing mice and cancer patients. However, the mechanisms by which ImC suppress immune responses in tumor-bearing mice have not been completely elucidated. In the present study, we investigated the function of splenic ImC freshly isolated from tumor-bearing mice and splenic ImC differentiated in vitro by GM-CSF. Freshly isolated splenic ImC were divided into two groups depending on Gr-1 expression, Gr-1 high (Gr-1^hi) and intermediate (Gr-1^int). Freshly isolated splenic Gr-1^int ImC, but not Gr-1^hi ImC, from tumor-bearing mice reduced production of IFN-γ in CD8⁺ T cells, but neither splenic Gr-1^int ImC nor Gr-1^hi ImC isolated from naive mice did. Both Gr-1^int and Gr-1^hi ImC differentiated in vitro by GM-CSF inhibited production of IFN-γ in both CD8⁺ and CD4⁺ T cells. In addition, the differentiated Gr-1^int ImC, one-third of which were CD11c⁺F4/80⁺ cells, and their culture supernatants suppressed proliferative responses of T cells stimulated by CD3 ligation, but the differentiated Gr-1^hi ImC and their culture supernatants did not. These results suggest that Gr-1^int ImC are altered to immune-suppressive cells in tumor circumstances and that they are differentiated by GM-CSF progressively into CD11c⁺F4/80⁺ cells with further suppressive activity against T cells.     

Fine mapping of the FecL locus influencing prolificacy in Lacaune sheep

In the Lacaune sheep population, two major loci influencing ovulation rate are segregating: FecX and FecL . The FecX ^L mutation is a non-conservative substitution (p.Cys53Tyr) in BMP15 that prevents the processing of the protein. Using a statistical approach, FecL has been shown to be an autosomal major gene. A full genome scan localized the FecL locus on sheep chromosome 11. Fine mapping reduced the interval containing FecL to markers BM17132 and FAM117A , corresponding to a synteny block of 1.1 megabases on human chromosome 17, which encompasses 20 genes. The expression of 16 genes from this interval was observed in tissues of the reproductive axis, but expression was not affected in homozygous FecL ^L females. In this interval, a unique haplotype was associated with the FecL ^L mutation. This particular haplotype could be predicted by the DLX3 :c.*803A>G SNP in the 3' UTR sequence of the DLX3 gene. This SNP provided accurate classification of animals (99.5%) as carriers or non-carriers of the mutation and therefore maybe useful in marker assisted selection. A synergistic action of FecL ^L and FecX ^L mutations on both ovulation rate and litter size was demonstrated. Until now, all the Fec genes identified in sheep belong to the bone morphogenetic protein (BMP) system. Based on the human orthologous region, none of the 20 genes in the FecL region corresponds to known molecules in the BMP system. The identification of the FecL ^L mutation could lead to the discovery of a new pathway involved in the regulation of ovulation rate.     

Identification and characterization of Bacillus anthracis by multiplex PCR analysis of sequences on plasmids pXO1 and pXO2 and chromosomal DNA

Abstract Bacillus anthracis can be identified on the basis of the detection of virulence factor genes located on two plasmids, pXO1 and pXO2. Thus isolates lacking both pXO1 and pXO2 are indistinguishable from closely related B. cereus group bacteria. We developed a multiplex PCR assay for characterization of B. anthracis isolates, and simultaneous confirmation of the species identity independent of plasmid content. The assay amplifies lef, cya, pag (pXO1) and cap (pXO2) genes, and a B. anthracis specific chromosomal marker, giving an easy-to-read profile. This system unambiguously identified virulent (pXO1⁺/2⁺) and avirulent (pXO1⁺/2⁻, pXO1⁻/2⁺ and pXO1⁻/2⁻) strains of B. anthracis and distinguished 'anthrax-like' strains from other B. cereus group bacteria.     

Differential Expression of Phospholipase C Specific for Inositol Phospholipids at the Cell Surface of Rat Glial Cells and REF52 Rat Embryo Fibroblasts

Abstract: Phosphatidylinositol(PI)-specific phospholipase C activity was detected on the surface of rat astrocytes, rat C6 glioma cells, and rat embryo (REF52) fibroblasts. The cell surface phospholipase C (ecto-PLC) activity was calcium-dependent, did not result from secreted phopholipase C, and was not released from the cell surface by bacterial PI-specific phospholipase C. Agents known to stimulate intracellular PI turnover, including carbachol, L-glutamic acid, acetylcholine, and orthovanadate, did not induce measurable alterations in the activity of the ecto-PLC. The expression of ecto-PLC activity by REF52 fibroblasts was density-dependent: subconfluent cultures of REF52 exhibited low levels of activity (less than 80 pmol of inositol phosphate formed/min/10⁶ cells), whereas in confluent cultures ecto-PLC activity increased to approximately 300 pmol/min/10⁶ cells. In contrast to this behavior and that exhibited by previously reported ecto-PLC-positive cell types, the ecto-PLC activity exhibited by astrocytes (approximately 1,000 pmol/min/10⁶ cells) and by C6 glioma cells (approximately 100 pmol/min/10⁶ cells) was independent of cell culture density up to confluence. The constitutive expression of ecto-PLC activity of astroglial cells may be related to their function as accessory cells in close association with neurons.     

Change of specific T cells in an emerging neonatal infectious disease induced by a bacterial superantigen

A new epidemic, NTED, has recently occurred in Japan. The cause of NTED is a bacterial superantigen, TSST-1. The aim of the present study was to analyze the change in Vβ2⁺ T cells reactive to TSST-1 in NTED in order to establish T-cell-targeted diagnostic criteria for NTED. Blood samples from 75 patients with clinically diagnosed NTED were collected from 13 neonatal intensive care units throughout Japan. We investigated the percentages of Vβ2⁺, Vβ3⁺ and Vβ12⁺ T cells and their CD45RO expressions in the samples using flow cytometry. In 18 of the 75 patients, we conducted multiple examinations of the T cells and monitored serial changes. The Vβ2⁺ T-cell population rapidly changed over three phases of the disease. Whereas the percentage of Vβ2⁺ T cells was widely distributed over the entire control range, CD45RO expression on Vβ2⁺ T cells in CD4⁺ in all 75 patients was consistently higher than the control range. Patients cannot necessarily be diagnosed as having NTED based on expansion of Vβ2⁺ T cells alone in the early acute phase. Instead, CD45RO expression on specific Vβ2⁺ cells is a potential diagnostic marker for a rapid diagnosis of NTED. We present three diagnostic categories of NTED. Fifty patients (66.7%) were included in the category 'definitive NTED'. It is important to demonstrate an increase of Vβ2⁺ T cells in the following phase in cases of 'probable NTED' or 'possible NTED'.     

Instability of the L6 gene for rust resistance in flax is correlated with the presence of a linked Ac element

In a programme aimed at tagging rust-resistance genes in flax with the maize transposable element Ac , a primary transformant of a line called 'Forge' that is homozygous for four rust-resistance genes, L ⁶, M, N and P ², was identified that possessed 10 copies of the Ac element, one of which was linked (29 map units) to L ⁶. Descendants of this plant, which had from 8 to 15 copies of Ac , were crossed to a rust-susceptible line and the progeny screened for rust-susceptible mutants. When the Ac linked to L ⁶ was present in the parent, a high frequency of L ⁶ mutants was observed (29 mutants in 30 575). By contrast, when this Ac was absent, no such mutants were observed in 9258 progeny. The background frequency of L ⁶ mutants was low (five in 124 088). A detailed analysis was made of the first 11 L ⁶ mutants recovered from parents carrying the L ⁶-linked Ac element. While none of the mutants possessed a tagged resistance gene, all lacked an RFLP marker closely linked to L ⁶, suggesting that deletions were responsible for loss of the L ⁶ specificity. In many of the mutants, one or more RFLP markers in the vicinity of the linked Ac were also absent. These findings suggest that the linked Ac may be inducing chromosome breakage.     

Osmoregulation of fission yeast: cloning of two distinct genes encoding glycerol-3-phosphate dehydrogenase, one of which is responsible for osmotolerance for growth

Many types of microorganisms, including both prokaryotes and eukaryotes, have developed mechanisms to adapt to severe osmotic stress. In this study, we isolated multicopy suppressor genes for a Schizosaccharomyces pombe mutant, which exhibited the clear phenotype of being osmosensitive for growth (Osm^s) on agar plates containing high concentrations of either non-ionic or ionic osmotic solutes. Two genes were thus identified, and each was suggested to encode an NADH-dependent glycerol-3-phosphate dehydrogenase (GPD), which is required for glycerol synthesis. The nucleotide sequences, determined for these genes (named gpd1 ⁺ and gpd2 ⁺, respectively), revealed that S. pombe has two distinct GPD isozymes. They are only 60% identical to each other in their amino acid sequences. One such isozyme, GPD1, was shown to be directly involved in osmoregulation, based on the following observations. (i) Expression of gpd1 ⁺ was regulated at the mRNA level in response to osmotic upshift, (ii) It was demonstrated that wild-type cells markedly accumulated internal glycerol under high-osmolarity growth conditions. (iii) Δ gpd1 mutants, however, failed to do so even in a high-osmolarity medium, and thus exhibited an Osm^s phenotype. On the other hand, the gpd2 ⁺ gene was constitutively expressed at a particular low level, regardless of the osmolarity of the medium.     

Resting and arecoline-stimulated brain metabolism and signaling involving arachidonic acid are altered in the cyclooxygenase-2 knockout mouse

Studies were performed to determine if cyclooxygenase (COX)-2 regulates muscarinic receptor-initiated signaling involving brain phospholipase A₂ (PLA₂) activation and arachidonic acid (AA; 20 : 4n-6) release. AA incorporation coefficients, k* (brain [1–¹⁴C]AA radioactivity/integrated plasma radioactivity), representing this signaling, were measured following the intravenous injection of [1–¹⁴C]AA using quantitative autoradiography, in each of 81 brain regions in unanesthetized COX-2 knockout (COX-2^–/–) and wild-type (COX-2^+/+) mice. Mice were administered arecoline (30 mg/kg i.p.), a non-specific muscarinic receptor agonist, or saline i.p. (baseline control). At baseline, COX-2^–/– compared with COX-2^+/+ mice had widespread and significant elevations of k*. Arecoline increased k* significantly in COX-2^+/+ mice compared with saline controls in 72 of 81 brain regions, but had no significant effect on k* in any region in COX-2^–/– mice. These findings, when related to net incorporation rates of AA from brain into plasma, demonstrate enhanced baseline brain metabolic loss of AA in COX-2^–/– compared with COX-2^+/+ mice, and an absence of a normal k* response to muscarinic receptor activation. This response likely reflects selective COX-2-mediated conversion of PLA₂-released AA to prostanoids.     

Sodium Transport from Blood to Brain: Inhibition by Furosemide and Amiloride

Abstract: Brain sodium uptake in vivo was studied using a modified intracarotid bolus injection technique in which the uptake of ²²Na ⁺ was compared with that of the relatively impermeable molecule, [³H]l-glucose. At a Na ⁺ concentration of 1.4 m M , Na ⁺ uptake was 1.74 ± 0.07 times greater than l -glucose uptake. This decreased to 1.34 ± 0.04 at 140 m M Na ⁺, indicating saturable Na ⁺ uptake. Relative Na ⁺ extraction was not affected by pH but was inhibited by amiloride ( K _i= 3 ± 10⁻⁷ M ) and by 1 m M furosemide. The effects of these two inhibitors were additive. Brain uptake of ⁸⁶Rb ⁺, a K ⁺ analogue, was measured to study interaction of K ⁺ with Na ⁺ transport systems. Relative ⁸⁶Rb ⁺ extraction was also inhibited by amiloride; however, it was not inhibited by furosemide. The results suggest the presence of two distinct transport systems that allow Na ⁺ to cross the luminal membrane of the brain capillary endothelial cell. These transport systems could play an important role in the movement of Na ⁺ from blood to brain.     

Natural competence in strains of Actinobacillus pleuropneumoniae

The fatty acid (FA) profiles of myxobacteria contain FA species with double bonds at the Δ⁵ and Δ¹¹ positions, the latter being rather unusual among bacteria. Despite this knowledge, the mechanism for introduction of these double bonds has never been described before in myxobacteria. Searches for candidate genes in the genome of the model organism Myxococcus xanthus revealed 16 genes, which have been annotated as FA desaturases. However, due to redundant substrate specificity, functional analyses of these enzymes by construction of inactivation mutants did not lead to the identification of their function or substrate specificity. Therefore, we elucidated the regioselectivity of the desaturation reactions by heterologous expression of eight desaturases from M. xanthus in Pseudomonas putida and thus could prove five of them to be indeed active as desaturases, with three (MXAN_1742, MXAN_3495 and MXAN_5461) and two (MXAN_0317 and MXAN_6306) acting as Δ⁵ and Δ¹¹ desaturases, respectively. This is the first report about the heterologous expression and regioselectivity of FA desaturases in myxobacteria.     

Genetic and environmental influences on self-reported and parent-reported behavior problems in young adult adoptees

The aim of the present study was to estimate the genetic, shared and nonshared environmental contributions to self-reported and parent-reported internalizing and externalizing problems in a follow-up study of intercountry adopted young adults. Young Adult Self-Report ratings were obtained from 1475 adoptees aged 22–32 years and Young Adult Behavior Checklist ratings from 1115 adoptive parents. For the genetic analyses, a subset of 143 adopted biologically related and 295 unrelated siblings was used. The data were subjected to model fitting decomposing three sources of variance: genetic factors (A) shared environment (C) and nonshared environment (E). Genetic factors were of more importance in both self-reported ( A ²= 54%, C ²= 0, and E ²= 46%) and parent-reported ( A ²= 76%, C ²= 15% and E ²= 9%) internalizing problems. Environmental factors were of more importance in both self-reported ( A ²= 33%, C ²= 17% and E ²= 50%) and parent-reported ( A ²= 28%, C ²= 27% and E ²= 45%) externalizing problems. This was in contrast with findings from the first and second assessments in the same sample during adolescence when genetic factors were more important in explaining externalizing problems compared with internalizing problems. Our results suggest a developmental reversal in genetic and environmental influences on behavior problems from early adolescence into adulthood, which could be related to different underlying developmental trajectories.