LacZ Activity Research Articles

Glucolipids and lipoteichoic acids affect the activity of SigI, an alternative sigma factor, and WalKR, an essential two-component system, in Bacillus subtilis.

In a Bacillus subtilis ugtP mutant lacking glucolipids, SigI was activated in the log phase, and the activation of SigI in the mutant was suppressed by the expression of native ugtP. By contrast, SigI was inhibited in a yfnI mutant lacking one of the lipoteichoic acid (LTA) synthase genes, and the inhibition was suppressed by the expression of yfnI. A series of mutation analyses of the sigI promoter revealed that the two WalR binding sites were involved in the increase of PsigI -lacZ activity in the ugtP mutant and decrease of the lacZ activity in the yfnI mutant. Transcription from the SigI recognition sequence was enhanced in the ugtP mutant, whereas yfnI disruption inhibited the transcription from the SigA recognition sequence in the sigI promoter. We found that not only SigI but also WalKR, the essential two-component system, was activated in the ugtP mutant and inhibited in the yfnI mutant. The walK mutants with activated WalR exhibited abnormal morphology, but this phenotype was suppressed by the addition of MgSO4 . We conclude that glucolipids and LTA are key compounds in the maintenance of normal cell surface structure in B. subtilis.

A dual prokaryotic (E. coli) expression system (pdMAX).

In this study, we introduced an efficient subcloning and expression system with two inducible prokaryotic expression promoters, arabinose and lac, in a single plasmid in Escherichia coli. The arabinose promoter unit allows for the expression of a FLAG-tagged protein, while the isopropyl-β-D-thiogalactoside (IPTG)-inducible unit allows for the expression of a Myc-tagged protein. An efficient subcloning (DNA insertion) system (iUnit) follows each promoter. The iUnit, based on a toxin that targets DNA topoisomerase of E. coli, allows for effective selection with arabinose or IPTG induction. With the dual promoter plasmid (pdMAX) system, expressed lacZ (β-galactosidase) activity was significantly decreased compared with the original solo expression system. Despite this disadvantage, we believe that the pdMAX system remains useful. A recombinant plasmid (pdMAX/ara/DsRed/IPTG/EGFP; pdMAX/DsRed/EGFP) with DsRed in the arabinose expression unit and EGFP in the IPTG expression unit showed fluorescent protein expression following additional low-temperature incubation. Thus, the novel pdMAX system allowed efficient subcloning of two different genes and can be used to induce and analyze the expression of two distinct genes. The proposed system can be applied to various types of prokaryotic gene expression analysis.

A dual prokaryotic (<i>E</i>.<i> coli</i>) expression system (pdMAX)

In this study, we introduced an efficient subcloning and expression system with two inducible prokaryotic expression promoters, arabinose and lac, in a single plasmid in Escherichia coli. The arabinose promoter unit allows for the expression of a FLAG-tagged protein, while the isopropyl-β-D-thiogalactoside (IPTG)-inducible unit allows for the expression of a Myc-tagged protein. An efficient subcloning (DNA insertion) system (iUnit) follows each promoter. The iUnit, based on a toxin that targets DNA topoisomerase of E. coli, allows for effective selection with arabinose or IPTG induction. With the dual promoter plasmid (pdMAX) system, expressed lacZ (β-galactosidase) activity was significantly decreased compared with the original solo expression system. Despite this disadvantage, we believe that the pdMAX system remains useful. A recombinant plasmid (pdMAX/ara/DsRed/IPTG/EGFP; pdMAX/DsRed/EGFP) with DsRed in the arabinose expression unit and EGFP in the IPTG expression unit showed fluorescent protein expression following additional low-temperature incubation. Thus, the novel pdMAX system allowed efficient subcloning of two different genes and can be used to induce and analyze the expression of two distinct genes. The proposed system can be applied to various types of prokaryotic gene expression analysis.

Localization and regulation of claudin-14 in experimental models of hypercalcemia.

Variations in the claudin-14 (CLDN14) gene have been linked to increased risk of hypercalciuria and kidney stone formation. However, the exact cellular localization of CLDN14 and its regulation remain to be fully delineated. To this end, we generated a novel antibody that allowed the detection of CLDN14 in paraffin-embedded renal sections. This showed CLDN14 to be detectable in the kidney only after induction of hypercalcemia in rodent models. Protein expression in the kidney is localized exclusively to the thick ascending limbs (TALs), mainly restricted to the cortical and upper medullary portion of the kidney. However, not all cells in the TALs expressed the tight junction protein. In fact, CLDN14 was primarily expressed in cells also expressing CLDN16 but devoid of CLDN10. CLDN14 appeared in very superficial apical cell domains and near cell junctions in a belt-like formation along the apical cell periphery. In transgenic mice, Cldn14 promotor-driven LacZ activity did not show complete colocalization with CLDN14 protein nor was it increased by hypercalcemia, suggesting that LacZ activity cannot be used as a marker for CLDN14 localization and regulation in this model. In conclusion, CLDN14 showed a restricted localization pattern in the apical domain of select cells of the TAL.

Retraction.

Retraction: Francis Domain, Xiaowen R. Bina and Stuart B. Levy, "Transketolase A, an enzyme in central metabolism, derepresses the marRAB multiple antibiotic resistance operon of Escherichia coli by interaction with MarR", Molecular Microbiology 2007, 66(2), 383-393 (https://doi/10.1111/j.1365-2958.2007.05928.x). The above article, published online on 14 September 2007 in Wiley Online Library (wileyonlinelibrary.com), has been retracted by agreement between the authors, the journal Editor in Chief John Helmann, and John Wiley & Sons Ltd. The retraction has been agreed because additional work in the authors' laboratory (L.M. McMurry) failed to confirm that the tktA gene overexpressed from pSPOK-TktA enhanced the expression in vivo of the mar promoter fusion marO-lacZ, or that deletion of chromosomal tktA reduced marO-lacZ expression. The original conclusions probably resulted from a yet unexplained variability of LacZ activity later discovered among different isolates of the marO-lacZ indicator strain SPC105. Moreover, the apparent binding of MarR to TktA-6H immobilized on NiNTA resin (Fig. 1B) was an artefact caused by non-specific binding of MarR alone to the resin that occurred when the wash contained no imidazole. Most MarR eluted in 20 mM imidazole without TktA-6H. Therefore, the conclusion that TktA interacts physically with MarR is not warranted. It is noted that Xiaowen R. Bina was not involved in work related to these errors. Note that previously a corrigendum was published to note these concerns (Molecular Microbiology (2011) 80(3), 853).

Quantitative analysis of the binding between the master bacterial iron regulator and a target DNA sequence

Iron is an essential micronutrient that is utilized by bacteria for numerous cellular and metabolic processes. The main way bacteria use to regulate iron homeostasis is via transcription regulation mediated by the ferric uptake regulator (Fur). In the presence of excess iron, the Fur protein acts as a transcriptional repressor of genes necessary for iron regulation. When the Fur protein binds to Fe2+, the Fur‐Fe2+ complex will bind to a target DNA sequence named the Fur box, which is located within the promoter of Fur‐regulated genes. However, when the iron level is limited, Fur dissociates from the Fur box and allows gene transcription. The Fur system, which is highly conserved throughout the different genera of bacteria, is absent in eukaryotes. Therefore, this unique feature of the Fur system combined with the essential nature of metal homeostasis in bacteria makes Fur a promising target for antimicrobials.Previous lab results identified a set of highly conserved residues in the Klebsiella sp. Fur protein, and site‐directed mutagenesis was carried out to obtain point mutants. β‐galactosidase assays were conducted to determine which mutants were affected in the in vivo and in vitro interaction between the Fur protein and a target DNA sequence (Fur box). Three mutants, H88A, H90A, and C96S, were found to exhibit a significantly higher level of LacZ activity, indicating that these are critical residues that affect the binding affinity to the DNA.In this work, the three mutant Fur proteins were overexpressed in E. coli BL21 cells and purified via affinity chromatography. A GST‐Fur protein was first purified using a GSH‐Sepharose resin, followed by cleavage of the GST tag and an additional purification step on a Heparin‐Sepharose resin. SDS‐PAGE analysis was used to identify fractions containing the purified recombinant proteins. In order to further examine the binding between Fur and the target DNA sequence, our lab applied BLItz biolayer interferometry. BLItz system is a direct binding assay that takes place on a biosensor probe. The biosensor surface only detects the molecules that bind directly to the probe, allowing us to determine the binding specificity and the dissociation constant (Kd). We obtained a commercially available streptavidin biosensor, which has a strong affinity for biotin‐labeled DNA (biotin‐labeled Fur box sequence). This allowed us to measure the binding constant between the Fur box and Fur protein by exposing the biotin‐labeled Fur box‐bound biosensor to the Fur protein.Among the wild‐type (WT), H88A, H90A, and C96S Fur proteins, the WT Fur protein showed significantly stronger binding affinity with the DNA compared to that of other three point‐mutant Fur proteins. These quantitative measures demonstrate that H88, H90, and C96 are indeed important residues that play major role in the binding affinity between the Fur protein and DNA binding.Support or Funding InformationThis project was supported by Midwestern University intramural funds. Samuel Cheong was a Kenneth A. Suarez summer research fellow at Midwestern University.

Broad-host-range application of the srfA promoter from Bacillus subtilis in Escherichia coli

The promoter of the srf operon (PsrfA) had been used to construct a cell-density-dependent expression system in B. subtilis in our previous work. The PsrfA and its derivative PsrfA12 showed good performance of heterologous protein expression in B. subtilis. In this work, using green fluorescent protein (GFP) and β-galactosidase (LacZ) as the reporter proteins, the host feasibility and expression characteristics of the PsrfA and PsrfA12 in E. coli were identified. The prominent green fluorescence shooted by laser scanning confocal microscope, fluorescence intensity measured by spectrophotometer and the distinct protein bands detected by SDS-PAGE demonstrated that the GFP could be largely expressed under the control of the PsrfA and PsrfA12 in the E. coli host strain of BL21 (DE3) and JM109 and the expression of GFP in strain BL21 (DE3) was much higher than that of in strain JM109. Meanwhile, the promoter PsrfA 12 was much stronger than PsrfA to the extent that the GFP controlled by PsrfA12 in strain BL21 (DE3) was leaked into the supernatant. And the fluorescence intensity detected in the supernatant of the recombinant strain BL21 (DE3) containing PsrfA12 was 10.25-fold higher than that of strain JM109 containing PsrfA. Moreover, the LacZ could also be produced by PsrfA and PsrfA12 in strain BL21 (DE3) and JM109 and the strain JM109 showed better performance than BL21 (DE3) in expressing LacZ. The LacZ activity controlled by PsrfA and PsrfA12 in JM109 were separately 2.47-fold and 2.36-fold higher than that of in strain BL21 (DE3). This work will broaden the applied range of the PsrfA and enrich the efficient toolbar for cross-species gene expression or module construction in E. coli and B. subtilis.

Effects of silver nanoparticles on pupation, eclosion, life span, apoptosis and protein expression in Drosophila melanogaster

Silver nanoparticle is widely used in the field of medicine because of its strong and effective antibacterial action. However, it has potential biological toxicity. In this study, the classical model organism, Drosophila melanogaster, was used to explore underlying mechanism for the toxic effects of silver nanoparticle. The pupation rate, eclosion time, eclosion rate and lifespan of Oregon R, w1118, and MTF mutants under different concentrations of silver nanoparticle were measured. The lacZ activity of rpr-lacZ strain was used to determine apoptosis of imaginal disc after treated with different concentrations of silver nanoparticle. The difference of intestinal protein expression in MTF mutants treated with different concentrations of silver nanoparticle was studied by SDS-PAGE. The amino acid sequence of differential proteins was further analyzed by mass spectrometry. The results showed that pupation rate and eclosion rate of MTF mutants significantly decreased when the concentration of silver nanoparticle increased to 200 μg·mL-1 and above. When the concentration of silver nanoparticle increased to 800 μg·mL-1, the rate of pupation and eclosion was significantly reduced, with the time of pupation and eclosion being not correlated to the concentration of silver nanoparticle. The concentrations of silver nanoparticle had no effect on the lifespan of Oregon R and w1118, while 200 μg·mL-1 silver nanoparticle significantly reduced the average lifespan of MTF mutant. Apoptosis increased with increasing concentration of silver nanoparticle. Results from SDS-PAGE and mass spectrometry analysis showed that the expression levels of proteins such as ATP kinase, heat shock protein and glucose metabolism related enzymes increased with increasing concentration of silver nanoparticle. Our results showed that high concentration of silver nanoparticle would reduce the survival rate of Drosophila, promote apoptosis and the expression of some proteins, which provided a theoretical basis for further understanding of the toxic mechanism of silver nanoparticle.

Enhanced Fluorescent Siderophore Biosynthesis and Loss of Phenazine-1-Carboxamide in Phenotypic Variant of Pseudomonas chlororaphis HT66.

Pseudomonas chlororaphis HT66 is a plant-beneficial bacterium that exhibits wider antagonistic spectrum against a variety of plant pathogenic fungi due to its main secondary metabolite, i.e., phenazine-1-carboxamide (PCN). In the present study, a spontaneous phenotypic variant designated as HT66-FLUO was isolated from the fermentation process of wild-type HT66 strain. The newly isolated phenotypic variant was morphologically distinct from the wild-type strain such as larger cell size, semi-transparent, non-production of PCN (Green or yellow crystals) and enhanced fluorescence under UV light. The whole-genome, RNA-sequencing, and phenotypic assays were performed to identify the reason of phenotypic variation in HT66-FLUO as compared to the HT66. Transcriptomic analysis revealed that 1,418 genes, representing approximately 22% of the 6393 open reading frames (ORFs) had undergone substantial reprogramming of gene expression in the HT66-FLUO. The whole-genome sequence indicated no gene alteration in HT66-FLUO as compared to HT66 according to the known reference sequence. The levels of global regulatory factor gacA and gacS expression were not significantly different between HT66 and HT66-FLUO. It was observed that overexpressing gacS rather than gacA in HT66-FLUO can recover switching of the variant to HT66. The β-galactosidase (LacZ) activity and qRT-PCR results indicate the downregulated expression of rsmX, rsmY, and rsmZ in HT66-FLUO as compared to HT66. Overexpressing three small RNAs in HT66-FLUO can revert switching of colony phenotype toward wild-type HT66 up to a certain degree, restore partial PCN production and reduces the fluorescent siderophores yield. However, the origin of the spontaneous phenotypic variant was difficult to be determined. In conclusion, this study helps to understand the gene regulatory effect in the spontaneous phenotypic variant.

A novel vector for transgenesis in the rat CNS

The larger brain of the rat enables a much greater repertoire of complex behaviors than mice, likely making rats preferential for investigating neurodegeneration. Because molecular tools for specific expression of transgenes in the rat brain are sparse, we chose Prnp encoding the prion protein (PrP) to develop a novel vector to drive transgene expression in the rat brain. We compared the rat Prnp sequence with mouse and Syrian hamster Prnp sequences, identifying conserved genetic elements and hypothesizing that these elements would be able to drive neuronal transgene expression. We investigated this by generating a vector termed RaPrnp that encompasses portions of the rat Prnp gene. Importantly, we replaced the rat Prnp open reading frame (ORF) with a cloning site for rapid and seamless In-Fusion cloning. To validate the in vivo neuronal specificity of the RaPrnp vector in rats, we generated stable RaPrnp-LacZ/enhanced green fluorescent protein (EGFP) transgenic (Tg) rat lines, which led to robust LacZ activity and high EGFP fluorescence in the central nervous system of embryos and adult animals. Next, we restored the rat Prnp ORF and generated multiple Tg(RaPrnp-PrP) lines, demonstrating that overexpression of Prnp accelerates the onset of scrapie. While the incubation time in wild-type (WT) rats was 175 ± 3 days post inoculation (dpi), one line, Tg2919, overexpressed RaPrPC at 4.4-fold and exhibited a reduced incubation time of 149 ± 2 dpi. The second line, Tg2922, overexpressed RaPrPC at 9.7-fold compared with WT animals and had an incubation time of 112 ± 0 dpi. Tg2922 rats inoculated with rat RML showed extensive vacuolation of the brainstem in contrast to WT and Tg2919 animals in which vacuolation was most prominent in the hippocampus and striatum as well as the motor and sensory cortices. It is possible that construction of Tg rats with modified phenotypes will prove more advantageous than mice for neurodegeneration studies.

Rapid and Scalable Preparation of Bacterial Lysates for Cell-Free Gene Expression.

Cell-free gene expression systems are emerging as an important platform for a diverse range of synthetic biology and biotechnology applications, including production of robust field-ready biosensors. Here, we combine programmed cellular autolysis with a freeze-thaw or freeze-dry cycle to create a practical, reproducible, and a labor- and cost-effective approach for rapid production of bacterial lysates for cell-free gene expression. Using this method, robust and highly active bacterial cell lysates can be produced without specialized equipment at a wide range of scales, making cell-free gene expression easily and broadly accessible. Moreover, live autolysis strain can be freeze-dried directly and subsequently lysed upon rehydration to produce active lysate. We demonstrate the utility of autolysates for synthetic biology by regulating protein production and degradation, implementing quorum sensing, and showing quantitative protection of linear DNA templates by GamS protein. To allow versatile and sensitive β-galactosidase (LacZ) based readout we produce autolysates with no detectable background LacZ activity and use them to produce sensitive mercury(II) biosensors with LacZ-mediated colorimetric and fluorescent outputs. The autolysis approach can facilitate wider adoption of cell-free technology for cell-free gene expression as well as other synthetic biology and biotechnology applications, such as metabolic engineering, natural product biosynthesis, or proteomics.

Bistability and Nonmonotonic Induction of the lac Operon in the Natural Lactose Uptake System

The Escherichia coli lac operon is regulated by a positive feedback loop whose potential to generate an all-or-none response in single cells has been a paradigm for bistable gene expression. However, so far bistable lac induction has only been observed using gratuitous inducers, raising the question about the biological relevance of bistable lac induction in the natural setting with lactose as the inducer. In fact, the existing experimental evidence points to a graded rather than an all-or-none response in the natural lactose uptake system. In contrast, predictions based on computational models of the lactose uptake pathway remain controversial. Although some argue in favor of bistability, others argue against it. Here, we reinvestigate lac operon expression in single cells using a combined experimental/modeling approach. To this end, we parameterize a well-supported mathematical model using transient measurements of LacZ activity upon induction with different amounts of lactose. The resulting model predicts a monostable induction curve for the wild-type system, but indicates that overexpression of the LacI repressor would drive the system into the bistable regime. Both predictions were confirmed experimentally supporting the view that the wild-type lac induction circuit generates a graded response rather than bistability. More interestingly, we find that the lac induction curve exhibits a pronounced maximum at intermediate lactose concentrations. Supported by our data, a model-based analysis suggests that the nonmonotonic response results from saturation of the LacI repressor at low inducer concentrations and dilution of Lac enzymes due to an increased growth rate beyond the saturation point. We speculate that the observed maximum in the lac expression level helps to save cellular resources by limiting Lac enzyme expression at high inducer concentrations.

Chloroform-free permeabilization for improved detection of β-galactosidase activity in Vibrio cholerae

LacZ (β-galactosidase) is used to monitor the transcription of genes in reporter strains carrying the lacZ gene under the control of a promotor of interest. This protocol for LacZ activity determinations in Vibrio cholerae following detergent lysis results in 2.5-fold increase of LacZ activities compared to lysis with chloroform.

A new method of producing NS5A antigen of hepatitis C virus

A task of creating a universal platform for engineering affordable recombinant producers of viral proteins conserving immunogenicity has not been solved yet. High toxicity of the viral proteins for the host cells, low yield and abnormal folding of the products often present severe obstacles to obtaining producers of the viral proteins. In this work, we report a new method of engineering and screening of deletion libraries from the viral antigen genes. This method allows selection of artificial derivatives of these genes adapted for expression in microbial producer cells. The method involves PCR amplification of the gene fragments using a system of randomized and adapter primers, which allows the spontaneous formation of duplexes from the random primers in the absence of the template DNA to be prevented. For selecting variants capable of in vivo expression, the obtained PCR products are cloned to a special vector of a direct phenotypical selection pQL30. It contains E. coli β-galactosidase gene with an inserted polylinker producing a frame-shift mutation. Using this screening method, an artificial variant of hepatitis C (HCV) NS5a gene with optimal biotechnological properties was established. 27 clinical specimens of 1670 bp long HCV1b NS5a fragments were used as a source gene. A PCR bank of the deletion derivatives was produced. 40 LacZ-positive clones based on pQL30 vector with a 50-700 bp long insertion were selected. The LacZ activity of the cell lysates and the immunogenicity of the products were tested. As a result, a single clone encoding a soluble protein with Mr = 114 kDa was selected. Its yield reached 0.3% of the total cell protein. It was highly reactive with sera of HCV 1b infected patients but not with sera of the healthy donors.

Editor's Highlight: Identification and Characterization of Teratogenic Chemicals Using Embryonic Stem Cells Isolated From a Wnt/β-Catenin-Reporter Transgenic Mouse Line.

Embryonic stem cells (ESCs) are commonly used for the analysis of gene function in embryonic development and provide valuable models for human diseases. In recent years, ESCs have also become an attractive tool for toxicological testing, in particular for the identification of teratogenic compounds. We have recently described a Bmp-reporter ESC line as a new tool to identify teratogenic compounds and to characterize the molecular mechanisms mediating embryonic toxicity. Here we describe the use of a Wnt/β-Catenin-reporter ESC line isolated from a previously described mouse line that carries the LacZ reporter gene under the control of a β-Catenin responsive promoter. The reporter ESC line stably differentiates into cardiomyocytes within 12 days. The reporter was endogenously induced between day 3-5 of differentiation reminiscent of its expression in vivo, in which strong LacZ activity is detected around gastrulation. Subsequently its expression becomes restricted to mesodermal cells and cells undergoing an epithelial to mesenchymal transition. The Wnt/β-Catenin-dependent expression of the reporter protein allowed quantification of dose- and time-dependent effects of teratogenic chemicals. In particular, valproic acid reduced reporter activity on day 7 whereas retinoic acid induced reporter activity on day 5 at concentrations comparable to the ones inhibiting the formation of functional cardiomyocytes, the classical read-out of the embryonic stem cell test (EST). In addition, we were also able to show distinct effects of teratogenic chemicals on the Wnt/β-Catenin-reporter compared with the previously described Bmp-reporter ESCs. Thus, different reporter cell lines provide complementary tools for the identification and analysis of potentially teratogenic compounds.

Expression of Evc2 in craniofacial tissues and craniofacial bone defects in Evc2 knockout mouse

ObjectiveOur objectives were to determine the expression of EVC2 in craniofacial tissues and investigate the effect of Evc2 deficiency on craniofacial bones using Evc2 knockout (KO) mouse model. DesignEvc2 KO mice were generated by introducing a premature stop codon followed by the Internal Ribosomal Entry Site fused to β-galactosidase (LacZ). Samples from wild-type (WT), heterozygous (Het) and homozygous Evc2 KO mice were prepared. LacZ staining and immunohistochemistry (IHC) with anti-β-galactosidase, anti-EVC2 and anti-SOX9 antibodies were performed. The craniofacial bones were stained with alcian blue and alizarin red. ResultsThe LacZ activity in KO was mainly observed in the anterior parts of viscerocranium. The Evc2-expressing cells were identified in many cartilageous regions by IHC with anti-β-galactosidase antibody in KO and Het embryos. The endogenous EVC2 protein was observed in these areas in WT embryos. Double labeling with anti-SOX9 antibody showed that these cells were mainly chondrocytes. At adult stages, the expression of EVC2 was found in chondrocytes of nasal bones and spheno-occipital synchondrosis, and osteocytes and endothelial-like cells of the premaxilla and mandible. The skeletal double staining demonstrated that craniofacial bones, where the expression of EVC2 was observed, in KO had the morphological defects as compared to WT. ConclusionTo our knowledge, our study was the first to identify the types of Evc2-expressing cells in craniofacial tissues. Consistent with the expression pattern, abnormal craniofacial bone morphology was found in the Evc2 KO mice, suggesting that EVC2 may be important during craniofacial growth and development.

Single-step method for β-galactosidase assays in Escherichia coli using a 96-well microplate reader

Historically, the lacZ gene is one of the most universally used reporters of gene expression in molecular biology. Its activity can be quantified using an artificial substrate, o-nitrophenyl-ß-d-galactopyranoside (ONPG). However, the traditional method for measuring LacZ activity (first described by J. H. Miller in 1972) can be challenging for a large number of samples, is prone to variability, and involves hazardous compounds for lysis (e.g., chloroform, toluene).Here we describe a single-step assay using a 96-well microplate reader with a proven alternative cell permeabilization method. This modified protocol reduces handling time by 90%.

Regulable Transgene Expression in Dorsal Root Ganglia of a Replication-Defective Herpes Simplex Virus Type 1 Vector by Means of Sciatic Nerve Injection.

Targeted and controllable gene delivery to neurons is essential to efforts to facilitate peripheral nerve regeneration. The authors investigated both the in vitro and in vivo expression profiles of a tetracycline-controlled, replication-defective, herpes simplex virus type 1-based vector. Mouse primary dorsal root ganglia cells were infected with QR9TO-LacZ in the absence or presence of tetracycline. LacZ gene expression was examined. It was also injected into sciatic nerves in CD-1 mice fed with and without tetracycline. LacZ expression in the upstream dorsal root ganglia was examined. Following inoculation with QR9TO-LacZ, approximately 40 percent of the cultured primary dorsal root ganglia cells exhibited strong LacZ activity in the presence of tetracycline at 48 and 72 hours, whereas little was detected in those in the absence of tetracycline. Quantitative analysis revealed that the β-galactosidase activity within cells exposed to tetracycline increased 181-fold at 48 hours (p < 0.001) and 47-fold at 72 hours after infection (p < 0.05) compared with those without tetracycline. However, this LacZ transgene activity in the presence of tetracycline tapered off to less than sevenfold over baseline 168 hours after infection (p < 0.05). Furthermore, successful uptake of this replication-defective viral vector was evident in upstream dorsal root ganglia after sciatic nerve injection in mice. In addition, its expression profile was similar to that in vitro, as strong β-galactosidase activity was evident only in mice fed with a doxycycline-containing diet, and it tapered off by 168 hours. The replication-defective herpes simplex virus type 1-based vector, which provides tightly regulated transgene expression in dorsal root ganglia by means of peripheral nerve injection, represents an appealing approach to improve peripheral nerve regeneration.

Adiponectin is required for cardiac MEF2 activation during pressure overload induced hypertrophy

Cardiomyocyte (CM) hypertrophy and increased heart mass in response to pressure overload are associated with hyper-activation of the myocyte enhancer factor-2 (MEF2) family of transcriptional regulators, and concomitant initiation of the fetal gene program. Adiponectin, an adipokine that is reduced in individuals with obesity and diabetes, has been characterized both as a negative regulator or permissive factor in cardiac hypertrophy. We therefore sought to analyze temporal regulation of MEF2 activity in response to pressure overload (PO) and changes in adiponectin status. To address this we crossed a well characterized transgenic MEF2 “sensor” mouse (MEF2-lacZ) with adiponectin null mice (Ad-KO) to create compound MEF2 lacZ/Ad-KO mice. Initially, we established that transverse aortic banding induced PO in wild-type (WT) mice increased heart mass and CM hypertrophy from 1 to 4weeks following surgery, indicated by increased CM diameter and heart weight/tibia length ratio. This was associated with cardiac dysfunction determined by echocardiography. Hypertrophic changes and dysfunction were observed in Ad-KO mice 4weeks following surgery. MEF2 lacZ activity and endogenous ANF mRNA levels, used as indicators of hypertrophic gene activation, were both robustly increased in WT mice after MTAB but attenuated in the Ad-KO background. Furthermore, activation of the pro-hypertrophic molecule p38 was increased following MTAB surgery in WT mice, but not in Ad-KO animals, and treatment of primary isolated CM with recombinant adiponectin induced p38 phosphorylation in a time dependent manner. Adiponectin also increased MEF2 activation in primary cardiomyocytes, an effect attenuated by p38 MAPK inhibition. In conclusion, our data indicate that robust hypertrophic MEF2 activation in the heart in vivo requires a background of adiponectin signaling and that adiponectin signaling in primary isolated CM directly enhances MEF2 activity through activation of p38 MAPK. We conclude that adiponectin is required for full induction of cardiomyocyte MEF2 activation, thus contributing to the myocardial hypertrophic gene expression program in response to PO.

The Origin of Mutants Under Selection: How Natural Selection Mimics Mutagenesis (Adaptive Mutation).

Selection detects mutants but does not cause mutations. Contrary to this dictum, Cairns and Foster plated a leaky lac mutant of Escherichia coli on lactose medium and saw revertant (Lac(+)) colonies accumulate with time above a nongrowing lawn. This result suggested that bacteria might mutagenize their own genome when growth is blocked. However, this conclusion is suspect in the light of recent evidence that revertant colonies are initiated by preexisting cells with multiple copies the conjugative F'lac plasmid, which carries the lac mutation. Some plated cells have multiple copies of the simple F'lac plasmid. This provides sufficient LacZ activity to support plasmid replication but not cell division. In nongrowing cells, repeated plasmid replication increases the likelihood of a reversion event. Reversion to lac(+) triggers exponential cell growth leading to a stable Lac(+) revertant colony. In 10% of these plated cells, the high-copy plasmid includes an internal tandem lac duplication, which provides even more LacZ activity—sufficient to support slow growth and formation of an unstable Lac(+) colony. Cells with multiple copies of the F'lac plasmid have an increased mutation rate, because the plasmid encodes the error-prone (mutagenic) DNA polymerase, DinB. Without DinB, unstable and stable Lac(+) revertant types form in equal numbers and both types arise with no mutagenesis. Amplification and selection are central to behavior of the Cairns-Foster system, whereas mutagenesis is a system-specific side effect or artifact caused by coamplification of dinB with lac. Study of this system has revealed several broadly applicable principles. In all populations, gene duplications are frequent stable genetic polymorphisms, common near-neutral mutant alleles can gain a positive phenotype when amplified under selection, and natural selection can operate without cell division when variability is generated by overreplication of local genome subregions.