Specificity of spontaneous mutations induced in mutA mutator cells

Escherichia coli cells expressing the mutA allele of a glyV (glycine tRNA) gene express a strong mutator phenotype. The mutA allele differs from the wild type glyV gene by a base substitution in the anticodon such that the resulting tRNA misreads certain aspartate codons as glycine, resulting in random, low-level Asp-->Gly substitutions in proteins. Subsequent work showed that many types of mistranslation can lead to a very similar phenotype, named TSM for translational stress-induced mutagenesis. Here, we have determined the specificity of forward mutations occurring in the lacI gene in mutA cells as well as in wild type cells. Our results show that in comparison to wild type cells, base substitutions are elevated 23-fold in mutA cells, as against a eight-fold increase in insertions and a five-fold increase in deletions. Among base substitutions, transitions are elevated 13-fold, with both G:C-->A:T and A:T-->G:C mutations showing roughly similar increases. Transversions are elevated 35-fold, with G:C-->T:A, G:C-->C:G and A:T-->C:G elevated 28-, 13- and 27-fold, respectively. A:T-->T:A mutations increase a striking 348-fold over parental cells, with most occurring at two hotspot sequences that share the G:C-rich sequence 5'-CCGCGTGG. The increase in transversion mutations is similar to that observed in cells defective for dnaQ, the gene encoding the proofreading function of DNA polymerase III. In particular, the relative proportions and sites of occurrence of A:T-->T:A transversions are similar in mutA and mutD5 (an allele of dnaQ) cells. Interestingly, transversions are also the predominant base substitutions induced in dnaE173 cells in which a missense mutation in the alpha subunit of polymerase III abolishes proofreading without affecting the 3'-->5' exonuclease activity of the epsilon subunit.     

Salutary effects of estrogen receptor-beta agonist on lung injury after trauma-hemorrhage

Although 17beta-estradiol (E2) administration after trauma-hemorrhage attenuates lung injury in male rodents, it is not known whether the salutary effects are mediated via estrogen receptor (ER)-alpha or ER-beta. We hypothesized that the salutary effects of E2 lung are mediated via ER-beta. Male Sprague-Dawley rats underwent trauma-hemorrhage (mean blood pressure 40 mmHg for 90 min, then resuscitation). E2 (50 microg/kg), ER-alpha agonist propyl pyrazole triol (PPT; 5 microg/kg), ER-beta agonist diarylpropiolnitrile (DPN; 5 microg/kg), or vehicle (10% DMSO) was injected subcutaneously during resuscitation. At 24 h after trauma-hemorrhage or sham operation, bronchoalveolar fluid (BALF) was collected for protein concentration, LDH activity, and nitrate/nitrite and IL-6 levels. Moreover, lung tissue was used for inducible nitric oxide synthase (iNOS) mRNA/protein expression, nitrate/nitrite and IL-6 levels, and wet/dry weight ratio (n = 6 rats/group). One-way ANOVA and Tukey's test were used for statistical analysis. The results indicated that E2 downregulated lung iNOS expression after trauma-hemorrhage. Protein concentration, LDH activity, and nitrate/nitrite and IL-6 levels in BALF and nitrate/nitrite and IL-6 levels in the lung increased significantly after trauma-hemorrhage; however, administration of DPN but not PPT significantly improved all parameters. Moreover, DPN treatment attenuated trauma-hemorrhage-mediated increase in iNOS mRNA/protein expression in the lung. In contrast, no significant change in the above parameters was observed with PPT. Thus the salutary effects of E2 on attenuation of lung injury are mediated via ER-beta, and ER-beta-induced downregulation of iNOS likely plays a significant role in the DPN-mediated lung protection after trauma-hemorrhage.     

Maintenance of lung myeloperoxidase activity in proestrus females after trauma-hemorrhage: upregulation of heme oxygenase-1

Previous studies showed that females in the proestrus stage of the reproductive cycle maintain organ functions after trauma-hemorrhage. However, it remains unknown whether the female reproductive cycle is an important variable in the regulation of lung injury after trauma-hemorrhage and, if so, whether the effect is mediated via upregulation of heme oxygenase (HO)-1. To examine this, female Sprague-Dawley rats during diestrus, proestrus, estrus, and metestrus phases of the reproductive cycle or 14 days after ovariectomy underwent soft tissue trauma and then hemorrhage (mean blood pressure 40 mmHg for 90 min followed by fluid resuscitation). At 2 h after trauma-hemorrhage or sham operation, lung myeloperoxidase (MPO) activity and intercellular adhesion molecule (ICAM)-1, cytokine-induced neutrophil chemoattractant (CINC)-1, CINC-3, and HO-1 protein levels were measured. Plasma 17beta-estradiol concentration was also determined. The results indicated that trauma-hemorrhage increased lung MPO activity and ICAM-1, CINC-1, and CINC-3 levels in ovariectomized females. These parameters were found to be similar to sham-operated animals in proestrus female rats subjected to trauma-hemorrhage. Lung HO-1 protein level in proestrus females was increased significantly compared with female rats subjected to trauma-hemorrhage during diestrus, estrus, and metestrus phases of the reproductive cycle and ovariectomized rats. Furthermore, plasma 17beta-estradiol level was highest in proestrus females. Administration of the HO inhibitor chromium mesoporphyrin prevented the attenuation of shock-induced lung damage in proestrus females. Thus these findings suggest that the female reproductive cycle is an important variable in the regulation of lung injury following trauma-hemorrhage and that the protective effect in proestrus females is likely mediated via upregulation of HO-1.     

Androstenediol ameliorates alterations in immune cells cytokine production capacity in a two-hit model of trauma-hemorrhage and sepsis

Although administration of androstenediol (a metabolite of dehydroepiandrosterone) following trauma-hemorrhage (T-H) produces beneficial effects on inflammatory cytokines and organ function, it remains unknown whether this metabolite has any salutary effects in preventing alterations in immune cell cytokine production following a combined insult of T-H and sepsis. To examine this, male rats underwent laparotomy, hemorrhagic shock (mean BP 40 mmHg for 90 min) and resuscitation or sham operation. Androstenediol (1 mg/kg BW i.v.) or vehicle was administered at the end of resuscitation. Twenty hrs after T-H or sham operation, sepsis was induced by cecal ligation and puncture (CLP). Five hours thereafter, plasma cytokine levels and cytokine production of various immune cells were determined. In a separate set of experiments, survival was monitored for 10 days after the induction of sepsis. Administration of androstenediol markedly decreased plasma IL-6 and TNF-alpha levels following T-H and CLP. Furthermore, it prevented the increased production of IL-6 and TNF-alpha by Kupffer cells and alveolar macrophages and attenuated the decrease in IL-6 and TNF-alpha production by splenic macrophages; however, it had no significant effects on the depressed IL-6 and TNF-alpha production by PBMC following T-H and CLP. The depressed IL-2 and IFN-gamma production by splenocytes under those conditions was attenuated by the administration of androstenediol. Furthermore, survival rate following T-H and subsequent sepsis was improved by androstenediol treatment. Since androstenediol administration following T-H attenuated cytokine production and reduced mortality in a double-hit model of T-H and sepsis, this agent appears to be a novel and useful adjunct for maintaining the immune cell functions following T-H and for decreasing the mortality rate from subsequent susceptibility to sepsis.     

Myosin Motors Drive Long Range Alignment of Actin Filaments

The bulk alignment of actin filament sliding movement, powered by randomly oriented myosin molecules, has been observed and studied using an in vitro motility assay. The well established, actin filament gliding assay is a minimal experimental system for studying actomyosin motility. Here, we show that when the assay is performed at densities of actin filaments approaching those found in living cells, filament gliding takes up a preferred orientation. The oriented patterns of movement that we have observed extend over a length scale of 10–100 μm, similar to the size of a mammalian cell. We studied the process of filament alignment and found that it depends critically upon filament length and density. We developed a simple quantitative measure of filament sliding orientation and this enabled us to follow the time course of alignment and the formation and disappearance of oriented domains. Domains of oriented filaments formed spontaneously and were separated by distinct boundaries. The pattern of the domain structures changed on the time scale of several seconds and the collision of neighboring domains led to emergence of new patterns. Our results indicate that actin filament crowding may play an important role in structuring the leading edge of migrating cells. Filament alignment due to near-neighbor mechanical interactions can propagate over a length scale of several microns; much greater than the size of individual filaments and analogous to a log drive. Self-alignment of actin filaments may make an important contribution to cell polarity and provide a mechanism by which cell migration direction responds to chemical cues.     

Androstenediol administration after trauma-hemorrhage attenuates inflammatory response, reduces organ damage, and improves survival following sepsis

Although androstenediol (adiol or 5-androstene-3beta,17beta-diol), a metabolite of dehydroepiandrosterone (DHEA), has protective effects following trauma-hemorrhage (T-H), it remains unknown whether administration of adiol has any salutary effects on the inflammatory response and outcome following a combined insult of T-H and sepsis. Male rats underwent T-H shock [mean arterial pressure (MAP) 40 mmHg for 90 min] followed by resuscitation. Adiol (1 mg/kg body wt) or vehicle was administered at the end of resuscitation. Sepsis was induced by cecal ligation and puncture (CLP) at 20 h after T-H or sham operation. Five hours after CLP, plasma and tissue samples were analyzed for cytokines (IL-6 and IL-10), MPO, neutrophil chemotactic factor (CINC-3), and liver injury (alanine aminotransferase and lactate dehydrogenase). In another group of rats, the gangrenous cecum was removed at 10 h after CLP, the cavity was irrigated with warm saline and closed in layers, and mortality was recorded over 10 days. T-H followed by CLP produced a significant elevation in plasma IL-6 and IL-10 levels, enhanced neutrophil cell activation, and resulted in liver injury. Adiol administration prevented the increase in cytokine production, neutrophil cell activation, and attenuated liver injury. Moreover, rats subjected to the combined insult, receiving vehicle or adiol, had a 50% and 6% mortality, respectively. Since adiol administration suppresses proinflammatory cytokines, reduces liver damage, and decreases mortality after the combined insult of T-H and sepsis, this agent appears to be a novel adjunct to fluid resuscitation for decreasing T-H-induced septic complications and mortality.     

Evaluation of affymetrix severe acute respiratory syndrome resequencing GeneChips in characterization of the genomes of two strains of coronavirus infecting humans

Severe acute respiratory syndrome (SARS) was discovered during a recent global outbreak of atypical pneumonia. A number of immunologic and molecular studies of the clinical samples led to the conclusion that a novel coronavirus (SARS-CoV) was associated with the outbreak. Later, a SARS resequencing GeneChip was developed by Affymetrix to characterize the complete genome of SARS-CoV on a single GeneChip. The present study was carried out to evaluate the performance of SARS resequencing GeneChips. Two human SARS-CoV strains (CDC#200301157 and Urbani) were resequenced by the SARS GeneChips. Five overlapping PCR amplicons were generated for each strain and hybridized with these GeneChips. The successfully hybridized GeneChips generated nucleotide sequences of nearly complete genomes for the two SARS-CoV strains with an average call rate of 94.6%. Multiple alignments of nucleotide sequences obtained from SARS GeneChips and conventional sequencing revealed full concordance. Furthermore, the GeneChip-based analysis revealed no additional polymorphic sites. The results of this study suggest that GeneChip-based genome characterization is fast and reproducible. Thus, SARS resequencing GeneChips may be employed as an alternate tool to obtain genome sequences of SARS-CoV strains pathogenic for humans in order to further understand the transmission dynamics of these viruses.     

Comparative evaluation of possible ocular photochemical toxicity of fluoroquinolones meant for ocular use in experimental models

Fluoroquinolones (FQs) are extensively used in bacterial keratitis and other intraocular infections. Since eye is constantly exposed to light, incidence of ocular phototoxicity due to commonly used FQs is of great interest for their safe use. Phototoxicity of commonly used FQs (ciprofloxacin, lomefloxacin, pefloxacin, ofloxacin, sparfloxacin and gatifloxacin), has been evaluated by using HET-CAM-UV model (Photo Hen Egg Test-C Chorioallantoic Membrane model). This study was further extended by adding lomefloxacin dissolved in bovine vitreous (0.5 ml) on the chorioallantoic membrane (CAM). Using a standard scale, the phototoxic damage was assessed at different time intervals. Respective controls were kept in dark to distinguish the toxicity of the drugs per se. The results showed that the phototoxicity induced by lomefloxacin was very high followed by gatifloxacin and sparfloxacin and least for other drugs studied. Interestingly, lomefloxacin along with vitreous showed significantly low phototoxicity. This could be due to the antioxidant property of ascorbic acid present in the vitreous.     

Staphylococcus aureus IsdB is a hemoglobin receptor required for heme iron utilization

The pathogenesis of human infections caused by the gram-positive microbe Staphylococcus aureus has been previously shown to be reliant on the acquisition of iron from host hemoproteins. The iron-regulated surface determinant system (Isd) encodes a heme transport apparatus containing three cell wall-anchored proteins (IsdA, IsdB, and IsdH) that are exposed on the staphylococcal surface and hence have the potential to interact with human hemoproteins. Here we report that S. aureus can utilize the host hemoproteins hemoglobin and myoglobin, but not hemopexin, as iron sources for bacterial growth. We demonstrate that staphylococci capture hemoglobin on the bacterial surface via IsdB and that inactivation of isdB, but not isdA or isdH, significantly decreases hemoglobin binding to the staphylococcal cell wall and impairs the ability of S. aureus to utilize hemoglobin as an iron source. Stable-isotope-tracking experiments revealed removal of heme iron from hemoglobin and transport of this compound into staphylococci. Importantly, mutants lacking isdB, but not isdH, display a reduction in virulence in a murine model of abscess formation. Thus, IsdB-mediated scavenging of iron from hemoglobin represents an important virulence strategy for S. aureus replication in host tissues and for the establishment of persistent staphylococcal infections.