Cyclooxygenase-2 (COX-2) is a critical enzyme implicated in chronic inflammation-associated cancer development. Our studies have shown that the exposure of Beas-2B cells, a human bronchial epithelial cell line, to lung carcinogenic nickel compounds results in increased COX-2 expression. However, the signaling pathways leading to nickel-induced COX-2 expression are not well understood. In the current study, we found that the exposure of Beas-2B cells to nickel compounds resulted in the activation of both nuclear factor of activated T cell (NFAT) and nuclear factor-κB (NF-κB). The expression of COX-2 induced upon nickel exposure was inhibited by either a NFAT pharmacological inhibitor or the knockdown of NFAT3 by specific siRNA. We further found that the activation of NFAT and NF-κB was dependent on each other. Since our previous studies have shown that NF-κB activation is critical for nickel-induced COX-2 expression in Beas-2B cells exposed to nickel compounds under same experimental condition, we anticipate that there might be a cross-talk between the activation of NFAT and NF-κB for the COX-2 induction due to nickel exposure in Beas-2B cells. Furthermore, we showed that the scavenging of reactive oxygen species (ROS) by introduction of mitochondrial catalase inhibited the activation of both NFAT and NF-κB, and the induction of COX-2 due to nickel exposure. Taken together, our results defining the evidence showing a key role of the cross-talk between NFAT and NF-κB pathways in regulating nickel-induced COX-2 expression, further provide insight into the understanding of the molecular mechanisms linking nickel exposure to its lung carcinogenic effects.
Beas-2B cells; COX-2; nickel; NFAT; NF-κB; ROS
CD4 counts and viral loads are dynamic quantities that change with time in HIV-infected persons. Commonly used single summary measures, such as viral load set point or early CD4 count do not explicitly account for changes in viral load or CD4 counts or other features of the overall time course of these measures. However, the efficient use of all repeated measurements within each subject is often a challenge made more difficult by sparse and irregular sampling over time. Here we illustrate how functional principal component (FPC) analysis provides an effective statistical approach for exploiting the patterns in CD4 count and viral load data over time. The method is demonstrated using data from Kenyan women who acquired HIV-1 during follow-up in a high risk cohort and were subsequently followed prospectively from early infection. The FPC scores for each woman obtained by this method serve as informative summary statistics for the CD4-count and viral-load trajectories. Similar to baseline CD4 count or viral set point, the first FPC score can be interpreted as a single-value summary measure of an individual's overall CD4 count or viral load. However, unlike most single-value summaries of CD4-count or viral-load trajectories, the first FPC score summarizes the dynamics of these quantities and is seen to reveal specific features of the trajectories associated with mortality in this cohort. Moreover, FPC scores are shown to be a more powerful prognostic factor than other common summaries when used in survival analysis.
longitudinal data; functional principal components; CD4 counts; viral loads
To fit within the confines of the cell, bacterial chromosomes are highly condensed into a structure called the nucleoid. Despite the high degree of compaction in the nucleoid, the genome remains accessible to essential biological processes, such as replication and transcription. Here, we present the first high-resolution chromosome conformation capture-based molecular analysis of the spatial organization of the Escherichia coli nucleoid during rapid growth in rich medium and following an induced amino acid starvation that promotes the stringent response. Our analyses identify the presence of origin and terminus domains in exponentially growing cells. Moreover, we observe an increased number of interactions within the origin domain and significant clustering of SeqA-binding sequences, suggesting a role for SeqA in clustering of newly replicated chromosomes. By contrast, ‘histone-like’ protein (i.e. Fis, IHF and H-NS) -binding sites did not cluster, and their role in global nucleoid organization does not manifest through the mediation of chromosomal contacts. Finally, genes that were downregulated after induction of the stringent response were spatially clustered, indicating that transcription in E. coli occurs at transcription foci.
Type III phosphatidylinositol-4-kinase beta (PI4KIIIβ) was previously implicated in hepatitis C virus (HCV) replication by small interfering RNA (siRNA) depletion and was therefore proposed as a novel cellular target for the treatment of hepatitis C. Medicinal chemistry efforts identified highly selective PI4KIIIβ inhibitors that potently inhibited the replication of genotype 1a and 1b HCV replicons and genotype 2a virus in vitro. Replicon cells required more than 5 weeks to reach low levels of 3- to 5-fold resistance, suggesting a high resistance barrier to these cellular targets. Extensive in vitro profiling of the compounds revealed a role of PI4KIIIβ in lymphocyte proliferation. Previously proposed functions of PI4KIIIβ in insulin secretion and the regulation of several ion channels were not perturbed with these inhibitors. Moreover, PI4KIIIβ inhibitors were not generally cytotoxic as demonstrated across hundreds of cell lines and primary cells. However, an unexpected antiproliferative effect in lymphocytes precluded their further development for the treatment of hepatitis C.
Following acute-phase infection, activated T cells are terminated to achieve immune homeostasis, failure of which results in lymphoproliferative and autoimmune diseases. We report that sterile α- and heat armadillo-motif-containing protein (SARM), the most conserved Toll-like receptors adaptor, is proapoptotic during T-cell immune response. SARM expression is significantly reduced in natural killer (NK)/T lymphoma patients compared with healthy individuals, suggesting that decreased SARM supports NK/T-cell proliferation. T cells knocked down of SARM survived and proliferated more significantly compared with wild-type T cells following influenza infection in vivo. During activation of cytotoxic T cells, the SARM level fell before rising, correlating inversely with cell proliferation and subsequent T-cell clearance. SARM knockdown rescued T cells from both activation- and neglect-induced cell deaths. The mitochondria-localized SARM triggers intrinsic apoptosis by generating reactive oxygen species and depolarizing the mitochondrial potential. The proapoptotic function is attributable to the C-terminal sterile alpha motif and Toll/interleukin-1 receptor domains. Mechanistically, SARM mediates intrinsic apoptosis via B cell lymphoma-2 (Bcl-2) family members. SARM suppresses B cell lymphoma-extra large (Bcl-xL) and downregulates extracellular signal-regulated kinase phosphorylation, which are cell survival effectors. Overexpression of Bcl-xL and double knockout of Bcl-2 associated X protein and Bcl-2 homologous antagonist killer substantially reduced SARM-induced apoptosis. Collectively, we have shown how T-cell death following infection is mediated by SARM-induced intrinsic apoptosis, which is crucial for T-cell homeostasis.
intrinsic T-cell death by SARM; influenza infection; adoptive transfer mouse model; neglect- and activation-induced cell death; NK/T-cell lymphoma
We report on the interfacial magnetic coupling in manganite bilayers of collinear ferromagnetic La0.7Sr0.3MnO3 and noncollinear multiferroic TbMnO3. Exchange bias is observed at the Néel temperature of TbMnO3 (~41 K) due to the onset of long-range antiferromagnetic ordering in the Mn spin sublattice. Interestingly, an anomalous plateau of exchange bias emerges at the ordering temperature of Tb spins (~10 K), and we ascribe this unique feature to the strong coupling between Tb and Mn spin sublattices in TbMnO3, which in turn influences the magnetic coupling across the interface. On the other hand, the enhancement of coercivity in La0.7Sr0.3MnO3/TbMnO3 shows monotonous temperature dependence. Our results illustrate a strong interfacial magnetic interaction at the La0.7Sr0.3MnO3/TbMnO3 interface, highlighting the roles of competing spin orders, magnetic frustration, and coupling between multiple spin sublattices in artificial collinear/noncollinear spin heterostructures.
We performed high resolution in vitro proton nuclear magnetic resonance spectroscopy on CSF and urine samples of 44 patients with leukodystrophies of unknown cause. Free sialic acid was elevated in CSF of two siblings with mental retardation and mild hypomyelination. By contrast, urinary excretion of free sialic acid in urine was normal on repeated testing by two independent methods. Both patients were homozygous for the K136E mutation in SLC17A5, the gene responsible for the free sialic acid storage diseases. Our findings demonstrate that mutations in the SLC17A5 gene have to be considered in patients with hypomyelination, even in the absence of sialuria.
Free Sialic Acid Storage Diseases; Leukodystrophy; NMR Spectroscopy
Coexistence of polarization and resistance-switching characteristics in single compounds has been long inspired scientific and technological interests. Here, we report the non-volatile resistance change in noncentrosymmetric compounds investigated by using defect nanotechnology and contact engineering. Using a noncentrosymmetric material of ZnO as example, we first transformed ZnO into high resistance state. Then ZnO electrical polarization was probed and its domains polarized 180° along the -axis with long-lasting memory effect (>25 hours). Based on our experimental observations, we have developed a vacancy-mediated pseudoferroelectricity model. Our first-principle calculations propose that vacancy defects initiate a spontaneous inverted domains nucleation at grain boundaries, and then they grow in the presence of an electrical field. The propagation of inverted domains follows the scanning tip motion under applied electrical field, leading to the growth of polarized domains over large areas.
The IRE1α-XBP1 pathway, a key component of the endoplasmic reticulum (ER) stress response, is considered to be a critical regulator for survival of multiple myeloma (MM) cells. Therefore, the availability of small-molecule inhibitors targeting this pathway would offer a new chemotherapeutic strategy for MM. Here, we screened small-molecule inhibitors of ER stress-induced XBP1 activation, and identified toyocamycin from a culture broth of an Actinomycete strain. Toyocamycin was shown to suppress thapsigargin-, tunicamycin- and 2-deoxyglucose-induced XBP1 mRNA splicing in HeLa cells without affecting activating transcription factor 6 (ATF6) and PKR-like ER kinase (PERK) activation. Furthermore, although toyocamycin was unable to inhibit IRE1α phosphorylation, it prevented IRE1α-induced XBP1 mRNA cleavage in vitro. Thus, toyocamycin is an inhibitor of IRE1α-induced XBP1 mRNA cleavage. Toyocamycin inhibited not only ER stress-induced but also constitutive activation of XBP1 expression in MM lines as well as primary samples from patients. It showed synergistic effects with bortezomib, and induced apoptosis of MM cells including bortezomib-resistant cells at nanomolar levels in a dose-dependent manner. It also inhibited growth of xenografts in an in vivo model of human MM. Taken together, our results suggest toyocamycin as a lead compound for developing anti-MM therapy and XBP1 as an appropriate molecular target for anti-MM therapy.
multiple myeloma; ER stress; IRE1α; XBP1; toyocamycin; adenosine analog
BACKGROUND AND AIMS
Although dietary fats and cholesterol have previously been associated with risk of cardiovascular disease (CVD) in middle aged populations, less is known among older adults. The purpose of this study was to determine the association between dietary fats, cholesterol, and eggs and CVD risk among community-dwelling adults aged 70–79 in the Health, Aging and Body Composition Study.
METHODS AND RESULTS
Diet was assessed using an interviewer-administered 108-item food frequency questionnaire (n=1,941). CVD events were defined as a confirmed myocardial infarction, coronary death, or stroke. Relative rates of CVD over 9 years of follow-up were estimated using Cox proportional hazards models. During follow-up, there were 203 incident cases of CVD. There were no significant associations between dietary fats and CVD risk. Dietary cholesterol (HR (95% CI): 1.47 (0.93, 2.32) for the upper vs. lower tertile; P for trend, 0.10) and egg consumption (HR (95% CI): 1.68 (1.12, 2.51) for 3+/week vs. <1/week); P for trend, 0.01) were associated with increased CVD risk. However, in subgroup analyses, dietary cholesterol and egg consumption were associated with increased CVD risk only among older adults with type 2 diabetes (HR (95% CI): 3.66 (1.09, 12.29) and 5.02 (1.63, 15.52), respectively, for the upper vs. lower tertile/group).
Dietary cholesterol and egg consumption were associated with increased CVD risk among older, community-dwelling adults with type 2 diabetes. Further research on the biological mechanism(s) for the increased CVD risk with higher dietary cholesterol and frequent egg consumption among older adults with diabetes is warranted.
The objective of this study was to provide insight into the molecular mechanisms of acute ischemic cerebrovascular syndrome (AICS) through gene expression profiling and pathway analysis.
Peripheral whole blood samples were collected from 39 MRI-diagnosed patients with AICS and 25 nonstroke control subjects ≥18 years of age. Total RNA was extracted from whole blood stabilized in Paxgene RNA tubes, amplified, and hybridized to Illumina HumanRef-8v2 bead chips. Gene expression was compared in a univariate manner between stroke patients and control subjects using t test in GeneSpring. The significant genes were tested in a logistic regression model controlling for age, hypertension, and dyslipidemia. Inflation of type 1 error was corrected by Bonferroni and Ingenuity Systems Pathway analysis was performed. Validation was performed by QRT-PCR using Taqman gene expression assays.
A 9-gene profile was identified in the whole blood of ischemic stroke patients using gene expression profiling. Five of these 9 genes were identified in a previously published expression profiling study of stroke and are therefore likely biomarkers of stroke. Pathway analysis revealed toll-like receptor signaling as a highly significant canonical pathway present in the peripheral whole blood of patients with AICS.
Our study highlights the relevance of the innate immune system through toll-like receptor signaling as a mediator of response to ischemic stroke and supports the claim that gene expression profiling can be used to identify biomarkers of ischemic stroke. Further studies are needed to validate and refine these biomarkers for their diagnostic potential.
= acute ischemic cerebrovascular syndrome;
= blood–brain barrier;
= Ingenuity Systems Pathway analysis;
= peripheral blood mononuclear cell;
= recombinant tissue plasminogen activator;
= toll-like receptor.
The Wnt gene family encodes a set of highly conserved secreted signaling proteins that have major roles in embryogenesis and tissue homeostasis. Yet the expression of this family of important mediators in psoriasis, a disease characterized by marked changes in keratinocyte growth and differentiation, is incompletely understood. We subjected 58 paired biopsies from lesional and uninvolved psoriatic skin and 64 biopsies from normal skin to global gene expression profiling. WNT5A transcripts were up-regulated 5-fold in lesional skin, accompanied by increased Wnt-5a protein levels. Notably, WNT5A mRNA was markedly induced by IL-1α, TNF-α, IFN-γ and TGF-α in cultured keratinocytes. FZD2 and FZD5, which encode receptors for Wnt5A, were also increased in lesional psoriatic skin. In contrast, expression of WIF1 mRNA, encoding a secreted antagonist of the Wnt proteins, was down-regulated >10-fold in lesional skin, along with decreased WIF-1 immunostaining. Interestingly, pathway analysis along with reduced AXIN2 expression and lack of nuclear translocation of beta-catenin indicated a suppression of canonical Wnt signaling in lesional skin.
Our results suggest a shift away from canonical Wnt signaling towards non-canonical pathways driven by interactions between Wnt-5a and its cognate receptors in psoriasis, accompanied by impaired homeostatic inhibition of Wnt signaling by WIF-1 and Dkk.
Wnt-signaling; psoriasis; WIF-1; Wnt proteins; keratinocytes
The wogonin-containing herb Scutellaria baicalensis has successfully been used for curing various diseases in traditional Chinese medicine. Wogonin has been shown to induce apoptosis in different cancer cells and to suppress growth of human cancer xenografts in vivo. However, its direct targets remain unknown. In this study, we demonstrate for the first time that wogonin and structurally related natural flavones, for example, apigenin, chrysin and luteolin, are inhibitors of cyclin-dependent kinase 9 (CDK9) and block phosphorylation of the carboxy-terminal domain of RNA polymerase II at Ser2. This effect leads to reduced RNA synthesis and subsequently rapid downregulation of the short-lived anti-apoptotic protein myeloid cell leukemia 1 (Mcl-1) resulting in apoptosis induction in cancer cells. We show that genetic inhibition of Mcl-1 or CDK9 expression by siRNA is sufficient to mimic flavone-induced apoptosis. Pull-down and in silico docking studies demonstrate that wogonin directly binds to CDK9, presumably to the ATP-binding pocket. In contrast, wogonin does not inhibit CDK2, CDK4 and CDK6 at doses that inhibit CDK9 activity. Furthermore, we show that wogonin preferentially inhibits CDK9 in malignant compared with normal lymphocytes. Thus, our study reveals a new mechanism of anti-cancer action of natural flavones and supports CDK9 as a therapeutic target in oncology.
anti-cancer drug; apoptosis; CDK9; Mcl-1; transcription
20S-protopanaxadiol (aPPD) is a metabolite of ginseng saponins, which is reported to be pro-apoptotic in some cells but anti-apoptotic in neuronal cells by regulating Akt signaling. Owing to its cholesterol-like structure, we hypothesized that aPPD may regulate Akt signaling by interacting with lipid rafts. Here, we compared Akt signaling in glioblastoma U87MG and neuroblastoma Neuro-2a cells treated with aPPD. aPPD did not change Akt activity in the total plasma membranes of each cell type, but drastically altered the activity of raft-associated Akt. Strikingly, Akt activity was decreased in the rafts of U87MG cells but increased in N2a cells by aPPD through regulating raft-associated dephosphorylation. The bidirectional regulation of raft-associated Akt signaling by aPPD enhanced the chemotoxicity of Paclitaxel or Vinblastine in U87MG cells but attenuated the excitotoxicity of N-methyl--aspartate in N2a cells. Our results demonstrated that the activity of raft-associated but not total membrane Akt determines its cellular functions. Lipid rafts differ in different types of cells, which allows for the possibility of cell-type-specific targeting for which aPPD might prove to be a useful agent.
20S-protopanaxadiol; Akt; apoptosis; lipid rafts
In order to identify new metabolic abnormalities in patients with complex neurodegenerative disorders of unknown aetiology, we performed high resolution in vitro proton nuclear magnetic resonance spectroscopy on patient cerebrospinal fluid (CSF) samples. We identified five adult patients, including two sisters, with significantly elevated free sialic acid in the CSF compared to both the cohort of patients with diseases of unknown aetiology (n = 144; P < 0.001) and a control group of patients with well-defined diseases (n = 91; P < 0.001). All five patients displayed cerebellar ataxia, with peripheral neuropathy and cognitive decline or noteworthy behavioural changes. Cerebral MRI showed mild to moderate cerebellar atrophy (5/5) as well as white matter abnormalities in the cerebellum including the peridentate region (4/5), and at the periventricular level (3/5). Two-dimensional gel analyses revealed significant hyposialylation of transferrin in CSF of all patients compared to age-matched controls (P < 0.001)—a finding not present in the CSF of patients with Salla disease, the most common free sialic acid storage disorder. Free sialic acid content was normal in patients’ urine and cultured fibroblasts as were plasma glycosylation patterns of transferrin. Analysis of the ganglioside profile in peripheral nerve biopsies of two out of five patients was also normal. Sequencing of four candidate genes in the free sialic acid biosynthetic pathway did not reveal any mutation. We therefore identified a new free sialic acid syndrome in which cerebellar ataxia is the leading symptom. The term CAFSA is suggested (cerebellar ataxia with free sialic acid).
cerebellar ataxia; free sialic acid; cerebrospinal fluid; neurometabolic disorder; nuclear magnetic resonance spectroscopy
In a Japanese study, cyclin-dependent kinase (CDK) based risk determined by CDK 1 and 2 activities was associated with risk of distance recurrence in early breast cancer patients. The aim of our study was to validate this risk categorization in European early breast cancer patients. We retrospectively analyzed frozen breast cancer specimens of 352 Dutch patients with histologically confirmed primary invasive early breast cancer. CDK-based risk was determined in tumour tissues by calculating a risk score (RS) according to kinases activity and protein mass concentration assay without the knowledge of outcome. Determination of CDK-based risk was feasible in 184 out of 352 (52%) tumours. Median follow-up of these patients was 15 years. In patients not receiving systemic treatment, the proportions of risk categories were 44% low, 16% intermediate, and 40% high CDK-based risk. These groups remained significant after univariate and multivariate Cox-regression analysis. Factors associated with a shorter distant recurrence-free period were positive lymph nodes, mastectomy with radiotherapy, and high CDK-based risk. There was no significant correlation with overall survival (OS). CDK-based risk is a prognostic marker of distance recurrence of patients with early breast cancer. More validation would be warranted to use of CDK-based risk into clinical practice.
breast cancer; cyclin-dependent kinase (CDK); distant recurrence prediction
Neurogenesis declines with advancing age. The mammalian achaete-scute homologue-1 encodes a basic helix–loop– helix transcription factor, which controls neuronal differentiation. In this study, we first tested whether atorvastatin treatment enhances neurological functional outcome and neuronal differentiation after stroke in retired breeder 12 month rats. Rats were subjected to middle cerebral artery occlusion and treated with or without atorvastatin (3 mg/kg) for 7 days. Atorvastatin significantly increased expression of mammalian achaete-scute homologue-1, β-tubulin III, and vascular endothelial growth factor in the ischemic brain, and concomitantly improved functional outcome compared with middle cerebral artery occlusion control rats. Increased neurogenesis significantly correlated with functional recovery after stroke. To further investigate the mechanisms of atorvastatin-induced neuronal differentiation, experiments were performed on neurospheres derived from retired breeder rat subventricular zone cells. Atorvastatin increased neuronal differentiation and upregulated vascular endothelial growth factor and mammalian achaete-scute homologue-1 gene expression in cultured neurospheres. Vascular endothelial growth factor–treated neurospheres significantly increased mammalian achaete-scute homologue-1 and β-tubulin III expression. Inhibition of vascular endothelial growth factor decreased atorvastatin-induced mammalian achaete-scute homologue-1 and β-tubulin III expression. These data indicate that atorvastatin increases neuronal differentiation in retired breeder rats. In addition, atorvastatin upregulation of vascular endothelial growth factor expression, influences mammalian achaete-scute homologue-1 transcription factor, which in turn, facilitates an increase in subventricular zone neuronal differentiation. These atorvastatin-mediated molecular events may contribute to the improved functional outcome in retired breeder rats subjected to stroke.
neuronal differentiation; Mash1; atorvastatin; VEGF; neurosphere; stroke
Recent reports have suggested that the Hedgehog (Hh) pathway is activated in lesional psoriatic skin, and that treatment with the Hh pathway antagonist cyclopamine may lead to rapid resolution of the disease. To assess Hh pathway activity in psoriasis, we isolated RNA from lesional and uninvolved skin of 58 psoriatic patients, and from 63 normal control subjects, and subjected these samples to global gene expression profiling on Affymetrix HU133 Plus 2.0 gene arrays. We were especially interested in Hh target genes (PTCH1, and GLI1), whose expression is elevated in response to Hh signaling. The microarray data demonstrated down-regulation of PTCH1 expression in uninvolved and lesional skin (1.1-fold and 2-fold respectively, p<0.0001). Additionally GLI1 mRNA was down-regulated in lesional skin (1.7 fold p<0.05). No significant changes were observed between lesional and uninvolved skin for the Hh ligands or Smoothened (SMO). QT-PCR confirmed these findings. In situ hybridization for GLI1 and PTCH1 was positive in BCC tumor cells, but was negligible in uninvolved or lesional psoriatic skin. The absence of elevated Hh target gene expression in lesional psoriatic skin indicates that the Hh pathway is not activated in this disease, raising questions regarding the proposed use of Hh antagonists as anti-psoriatic agents.
Psoriasis; GLI; PTCH1; sonic hedgehog pathway
Light information reaches the suprachiasmatic nucleus (SCN) through a subpopulation of retinal ganglion cells. Previous work raised the possibility that brain-derived neurotrophic factor (BDNF) and its high-affinity tropomyosin-related receptor kinase may be important as modulators of this excitatory input into the SCN. In order to test this possibility, we used whole-cell patch-clamp methods to measure spontaneous excitatory currents in mouse SCN neurons. We found that the amplitude and frequency of these currents were increased by BDNF and decreased by the neurotrophin receptor inhibitor K252a. The neurotrophin also increased the magnitude of currents evoked by application of N-methyl-D-aspartate and amino-methyl proprionic acid. Next, we measured the rhythms in action potential discharge from the SCN brain slice preparation. We found that application of K252a dramatically reduced the magnitude of phase shifts of the electrical activity rhythm generated by the application of glutamate. By itself, BDNF caused phase shifts that resembled those produced by glutamate and were blocked by K252a. The results demonstrate that BDNF and neurotrophin receptors can enhance glutamatergic synaptic transmission within a subset of SCN neurons and potentiate glutamate-induced phase shifts of the circadian rhythm of neural activity in the SCN.
circadian rhythms; mouse; neurotrophin; N-methyl-d-aspartate; suprachiasmatic nucleus
The Escherichia coli l-rhamnose-responsive transcription activators RhaS and RhaR both consist of two domains, a C-terminal DNA-binding domain and an N-terminal dimerization domain. Both function as dimers and only activate transcription in the presence of l-rhamnose. Here, we examined the ability of the DNA-binding domains of RhaS (RhaS-CTD) and RhaR (RhaR-CTD) to bind to DNA and activate transcription. RhaS-CTD and RhaR-CTD were both shown by DNase I footprinting to be capable of binding specifically to the appropriate DNA sites. In vivo as well as in vitro transcription assays showed that RhaS-CTD could activate transcription to high levels, whereas RhaR-CTD was capable of only very low levels of transcription activation. As expected, RhaS-CTD did not require the presence of l-rhamnose to activate transcription. The upstream half-site at rhaBAD and the downstream half-site at rhaT were found to be the strongest of the known RhaS half-sites, and a new putative RhaS half-site with comparable strength to known sites was identified. Given that cyclic AMP receptor protein (CRP), the second activator required for full rhaBAD expression, cannot activate rhaBAD expression in a ΔrhaS strain, it was of interest to test whether CRP could activate transcription in combination with RhaS-CTD. We found that RhaS-CTD allowed significant activation by CRP, both in vivo and in vitro, although full-length RhaS allowed somewhat greater CRP activation. We conclude that RhaS-CTD contains all of the determinants necessary for transcription activation by RhaS.
In contrast to eukaryotes, bacteria such as Escherichia coli contain only one form of RNA polymerase (RNAP), which is responsible for all cellular transcription. Using an RNAP-green fluorescent protein fusion protein, we showed previously that E. coli RNAP is partitioned exclusively in the nucleoid and that stable RNA synthesis, particularly rRNA transcription, is critical for concentrating a significant fraction of RNAP in transcription foci during exponential growth. The extent of focus formation varies under different physiological conditions, supporting the proposition that RNAP redistribution is an important element for global gene regulation. Here we show that extra, plasmid-borne copies of an rRNA operon recruit RNAP from the nucleoid into the cytoplasmic space and that this is accompanied by a reduction in the growth rate. Transcription of an intact rRNA operon is not necessary, although a minimal transcript length is required for this phenotype. Replacement of the ribosomal promoters with another strong promoter, Ptac, abolished the effect. These results demonstrate that active synthesis from rRNA promoters is a major driving force for the distribution of RNAP in bacteria. The implications of our results for the regulation of rRNA synthesis and cell growth are discussed.
Lipopolysaccharide (LPS), shed by gram-negative bacteria during infection and antimicrobial therapy, may lead to lethal endotoxic shock syndrome. A rational design strategy based on the presumed mechanism of antibacterial effect was adopted to design cationic antimicrobial peptides capable of binding to LPS through tandemly repeated sequences of alternating cationic and nonpolar residues. The peptides were designed to achieve enhanced antimicrobial potency due to initial bacterial membrane binding with a reduced risk of endotoxic shock. The peptides designed displayed binding affinities to LPS and lipid A (LA) in the low micromolar range and by molecular modeling were predicted to form amphipathic β-hairpin-like structures when they bind to LPS or LA. They also exhibited strong effects against gram-negative bacteria, with MICs in the nanomolar range, and low cytotoxic and hemolytic activities at concentrations significantly exceeding their MICs. Quantitative structure-activity relationship (QSAR) analysis of peptide sequences and their antimicrobial, cytotoxic, and hemolytic activities revealed that site-directed substitutions of residues in the hydrophobic face of the amphipathic peptides with less lipophilic residues selectively decrease the hemolytic effect without significantly affecting the antimicrobial or cytotoxic activity. On the other hand, the antimicrobial effect can be enhanced by substitutions in the polar face with more polar residues, which increase the amphipathicity of the peptide. On the basis of the QSARs, new analogs that have strong antimicrobial effects but that lack hemolytic activity can be proposed. The findings highlight the importance of peptide amphipathicity and allow a rational method that can be used to dissociate the antimicrobial and hemolytic effects of cationic peptides, which have potent antimicrobial properties, to be proposed.
An auxiliary β2 subunit, when coexpressed with Slo α subunits, produces inactivation of the resulting large-conductance, Ca2+ and voltage-dependent K+ (BK-type) channels. Inactivation is mediated by the cytosolic NH2 terminus of the β2 subunit. To understand the structural requirements for inactivation, we have done a mutational analysis of the role of the NH2 terminus in the inactivation process. The β2 NH2 terminus contains 46 residues thought to be cytosolic to the first transmembrane segment (TM1). Here, we address two issues. First, we define the key segment of residues that mediates inactivation. Second, we examine the role of the linker between the inactivation segment and TM1. The results show that the critical determinant for inactivation is an initial segment of three amino acids (residues 2–4: FIW) after the initiation methionine. Deletions that scan positions from residue 5 through residue 36 alter inactivation, but do not abolish it. In contrast, deletion of FIW or combinations of point mutations within the FIW triplet abolish inactivation. Mutational analysis of the three initial residues argues that inactivation does not result from a well-defined structure formed by this epitope. Inactivation may be better explained by linear entry of the NH2-terminal peptide segment into the permeation pathway with residue hydrophobicity and size influencing the onset and recovery from inactivation. Examination of the ability of artificial, polymeric linkers to support inactivation suggests that a variety of amino acid sequences can serve as adequate linkers as long as they contain a minimum of 12 residues between the first transmembrane segment and the FIW triplet. Thus, neither a specific distribution of charge on the linker nor a specific structure in the linker is required to support the inactivation process.
inactivation mechanisms; inactivation domains; K+ channels; BK channels; Ca2+- and voltage-gated K+ channels
An unusual case of retroperitoneal ectopic pregnancy is reported. The patient, a 34 year old nulligravida, underwent IVF/ET following bilateral salpingectomy. A small, degenerating, intrauterine gestational sac suggested failing intrauterine pregnancy. There was no intraperitoneal free fluid. On Day 41 after ET, the patient was hospitalized because of acute epigastric pains. A pseudocyst of the head of pancreas was demonstrated by CT scan. A day later, exploratory laparotomy, because of a precipitous drop in the hemoglobin, revealed a massive retroperitoneal hematoma and an embryo in the gestational sac attached to the head of pancreas and major blood vessels. The patient did well following surgery. The mechanisms of retroperitoneal embryo migration are discussed and literature is reviewed.
Ectopic pregnancy; IVF; bilateral salpingectomy; retroperitoneal upper abdominal pregnancy
A family of auxiliary β subunits coassemble with Slo α subunit to form Ca2+-regulated, voltage-activated BK-type K+ channels. The β subunits play an important role in regulating the functional properties of the resulting channel protein, including apparent Ca2+ dependence and inactivation. The β3b auxiliary subunit, when coexpressed with the Slo α subunit, results in a particularly rapid (∼1 ms), but incomplete inactivation, mediated by the cytosolic NH2 terminus of the β3b subunit (Xia et al. 2000). Here, we evaluate whether a simple block of the open channel by the NH2-terminal domain accounts for the inactivation mechanism. Analysis of the onset of block, recovery from block, time-dependent changes in the shape of instantaneous current-voltage curves, and properties of deactivation tails suggest that a simple, one step blocking reaction is insufficient to explain the observed currents. Rather, blockade can be largely accounted for by a two-step blocking mechanism () in which preblocked open states (O*n) precede blocked states (In). The transitions between O* and I are exceedingly rapid accounting for an almost instantaneous block or unblock of open channels observed with changes in potential. However, the macroscopic current relaxations are determined primarily by slower transitions between O and O*. We propose that the O to O* transition corresponds to binding of the NH2-terminal inactivation domain to a receptor site. Blockade of current subsequently reflects either additional movement of the NH2-terminal domain into a position that hinders ion permeation or a gating transition to a closed state induced by binding of the NH2 terminus.
channel block; K+ channels; gating mechanisms; Ca2+- and voltage-gated K+ channels; mSlo channels •