Levels Of Β-galactosidase Activity Research Articles

The impact of glutaredoxin 1 and glutaredoxin 2 double knockout on lens epithelial cell function

Glutaredoxins (Grx1 and Grx2) are thiol-repair antioxidant enzymes that play vital roles in cellular redox homeostasis and various cellular processes. This study aims to evaluate the functions of the glutaredoxin (Grx) system, including glutaredoxin 1 (Grx1) and glutaredoxin 2 (Grx2), using Grx1/Grx2 double knockout (DKO) mice as a model. We isolated primary lens epithelial cells (LECs) from wild-type (WT) and DKO mice for a series of in vitro analyses. Our results revealed that Grx1/Grx2 DKO LECs exhibited slower growth rates, reduced proliferation, and aberrant cell cycle distribution compared to WT cells. Elevated levels of β-galactosidase activity were observed in DKO cells, along with a lack of caspase 3 activation, suggesting that these cells may be undergoing senescence. Additionally, DKO LECs displayed compromised mitochondrial function, characterized by decreased ATP production, reduced expression levels of oxidative phosphorylation (OXPHOS) complexes III and IV, and increased proton leak. A compensatory metabolic shift towards glycolysis was observed in DKO cells, indicating an adaptive response to Grx1/Grx2 deficiency. Furthermore, loss of Grx1/Grx2 affected cellular structure, leading to increased polymerized tubulin, stress fiber formation, and vimentin expression in LECs. In conclusion, our study demonstrates that Grx1/Grx2 double deletion in LECs results in impaired cell proliferation, aberrant cell cycle progression, disrupted apoptosis, compromised mitochondrial function, and altered cytoskeletal organization. These findings underscore the importance of Grx1 and Grx2 in maintaining cellular redox homeostasis and the consequences of their deficiency on cellular structure and function. Further research is needed to elucidate the precise molecular mechanisms underlying these observations and to investigate potential therapeutic strategies targeting Grx1 and Grx2 for various physiological processes and oxidative-stress related diseases such as cataract.

Estrogen Signaling Induces Mitochondrial Dysfunction-Associated Autophagy and Senescence in Breast Cancer Cells.

Previous work has shown that although estrogen (E2) disrupts cellular iron metabolism and induces oxidative stress in breast and ovarian cancer cells, it fails to induce apoptosis. However, E2 treatment was reported to enhance the apoptotic effects of doxorubicin in cancer cells. This suggests that E2 can precipitate anti-growth effects that render cancer cells more susceptible to chemotherapy. To investigate such anti-growth non-apoptotic, effects of E2 in cancer cells, MDA-MB-231 and MCF-7 cells were evaluated for the expression of key autophagy and senescence markers and for mitochondrial damage following E2 treatment. Treated cells experienced mitochondrial membrane depolarization along with increased expression of LC3-I/II, Pink1 and LAMP2, increased LC3-II accumulation and increased lysosomal and mitochondrial accumulation and flattening. E2-treated MCF-7 cells also showed reduced P53 and pRb780 expression and increased Rb and P21 expression. Increased expression of the autophagy markers ATG3 and Beclin1 along with increased levels of β-galactosidase activity and IL-6 production were evident in E2-treated MCF-7 cells. These findings suggest that E2 precipitates a form of mitochondrial damage that leads to cell senescence and autophagy in breast cancer cells.

Identification and characterization of BoPUB3: a novel interaction protein with S-locus receptor kinase in Brassica oleracea L.

Armadillo repeat containing 1 (ARC1) is phosphorylated by S-locus receptor kinase (SRK) and functions as a positive regulator in self-incompatibility response of Brassica. However, ARC1 only causes partial breakdown of the self-incompatibility response, and other SRK downstream factors may also participate in the self-incompatibility signaling pathway. In the present study, to search for SRK downstream targets, a plant U-box protein 3 (BoPUB3) was identified from the stigma of Brassica oleracea L. BoPUB3 was highly expressed in the stigma, and its expression was increased with the stigma development and reached to the highest level in the mature-stage stigma. BoPUB3, a 76.8-kDa protein with 697 amino acids, is a member of the PUB-ARM family and contains three domain characteristics of BoARC1, including a U-box N-terminal domain, a U-box motif, and a C-terminal arm repeat domain. The phylogenic tree showed that BoPUB3 was close to BoARC1. The synteny analysis revealed that B. oleracea chromosomal region containing BoPUB3 had high synteny with the Arabidopsis thaliana chromosomal region containing AtPUB3 (At3G54790). In addition, the subcellular localization analysis showed that BoPUB3 primarily localized in the plasma membrane and also in the cytoplasm. The combination of the yeast two-hybrid and in vitro binding assay showed that both BoPUB3 and BoARC1 could interact with SRK kinase domain, and SRK showed much higher level of β-galactosidase activity in its interaction with BoPUB3 than with BoARC1. These results implied that BoPUB3 is a novel interactor with SRK, which lays a basis for further research on whether PUB3 participates in the self-incompatibility signaling pathway.

Construction of targeted and integrative promoter-reporter plasmids pDK-K and pDK-G to measure gene expression activity in Haemophilus parasuis

Haemophilus parasuis (H. parasuis) is rather difficult to manipulate genetically due to the diversity of restriction-modification systems and other mechanisms harbored by various isolates. This prevents exogenous plasmids from replicating in this species and hinders research efforts focused on transcriptional regulators in this bacterium. In this study, we generated a convenient promoter reporter system based on gene knock-in method using natural transformation in H. parasuis. Gene knock-in has proven useful as a powerful tool facilitating identification and studying the transcription activities of regulators under a variety of conditions that favor gene transcription or expression from an incorporated promoter. The vectors, pDK-K and pDK-G, carrying promoterless reporter lacZ gene and two homologous sequences flanking a knock-in site, may have some advantages over the extensively used plasmid-bearing reporter system in other bacteria in stability and ease of genetic manipulation in H. parasuis. The knock-in site was positioned at a site occupied by flanking genes that were both hypothetical and had the same transcription orientation, thus the expression of the reversely cloned promoter-lacZ fusion wouldn't be affected by the upstream promoter on the chromosome. The expression activity of lacZ gene under the transcriptional activation of a 300 bp promoter-proximal segment of cyaA, crp or comA genes in H. parasuis was separately validated using X-gal and o-nitrophenyl-β-d-galactoside(ONPG) as substrates. The derivatives harboring promoter-lacZ fusion segments showed significantly higher β-galactosidase activity levels than the promoterlessones both in TSB++ broth and on TSA++ plate as screened either by X-gal method or the standard Miller method. We also used pDK vector to further certify that the cyaA promoter is inducible and whose transcriptional levels were in correlation with the growth kinetics of the bacteria in TSB++. With this system, gene knock-in method based on natural transformation in H. parasuis proved to be useful in identifying transcriptional regulation of a certain promoter.

Regulation of Expression of Oxacillin-Inducible Methionine Sulfoxide Reductases in Staphylococcus aureus.

Cell wall-active antibiotics cause induction of a locus that leads to elevated synthesis of two methionine sulfoxide reductases (MsrA1 and MsrB) in Staphylococcus aureus. To understand the regulation of this locus, reporter strains were constructed by integrating a DNA fragment consisting of the msrA1/msrB promoter in front of a promoterless lacZ gene in the chromosome of wild-type and MsrA1-, MsrB-, MsrA1/MsrB-, and SigB-deficient methicillin-sensitive S. aureus strain SH1000 and methicillin-resistant S. aureus strain COL. These reporter strains were cultured in TSB and the cellular levels of β-galactosidase activity in these cultures were assayed during different growth phases. β-galactosidase activity assays demonstrated that the lack of MsrA1, MsrB, and SigB upregulated the msrA1/msrB promoter in S. aureus strain SH1000. In S. aureus strain COL, the highest level of β-galactosidase activity was observed under the conditions when both MsrA1 and MsrB proteins were absent. The data suggest that the msrA1/msrB locus, in part, is negatively regulated by MsrA1, MsrB, and SigB in S. aureus.

Altered expression pattern of Nrf2/HO-1 axis during accelerated-senescence in HIV-1 transgenic rat.

Chronic oxidative stress plays a central role in the pathogenesis of many diseases, including HIV-1 associated disorders. Concomitantly with the decline of endogenous antioxidant systems, it was reported that HIV-1-related proteins increase the production of radical species in cells and tissues that are not directly infected by the virus. In the context of HIV-1 infection, the role of Nrf2, a key transcription factor that contributes to the maintenance of cellular redox homeostasis, remains largely uncharacterized. One of the major stress-responsive player regulated by Nrf2 is the antioxidant enzyme HO-1. The Nrf2/HO-1 axis constitutes a crucial cell survival mechanism to counteract oxidative stress and inflammation. The present study aims to investigate the age-related patterns of Nrf2 and HO-1 in different brain regions and tissues of HIV-1 transgenic rat. Since HIV-1 induces an accelerated aging and the redox imbalance may actively promote senescence, we also evaluated the senescence phenotype-switching by quantifying levels of β-galactosidase activity. Our results showed changes in gene expression, with different trends depending on the brain regions and tissues examined. However, compared to age-matched controls, we observed in HIV-1 transgenic rats a significant reduction in the protein levels of Nrf2 and HO-1, suggesting a weakening in the protection exerted by Nrf2/HO-1 system. Moreover, we show that senescence occurs more rapidly in HIV-1 transgenic rats than in control animals. To our knowledge this is the first in vivo report showing the involvement of Nrf2/HO-1 pathway in a rat model of HIV-1.

A slow-release fibrin matrix increases adeno-associated virus transduction of wound repair cells in vivo

Virus-mediated gene therapy is a promising strategy for numerous tissue engineering applications. Fibrin-based scaffolds have been previously used as vehicles for localised delivery of adenovirus to wound sites. However, their utility in the delivery of adeno-associated viruses to wound repair cells has not yet been determined. The influence of fibrin concentration on efficacy of delivery of AAV-2 to wound tissue was assessed in this study. Fibrin scaffolds containing recombinant AAV-2 encoding for β-galactosidase were polymerised in porous polyurethane discs and implanted subcutaneously in rats. A fibrin scaffold with a concentration of 50 mg/ml showed significantly elevated levels of β-galactosidase activity within explanted discs at 10 days compared to 10 mg/ml and 25 mg/ml fibrin. These findings inform efforts to optimise biodegradable scaffolds for the localised delivery of AAV in tissue engineering.

OxyR-dependent expression of a novel glutathione S-transferase (Abgst01) gene in Acinetobacter baumannii DS002 and its role in biotransformation of organophosphate insecticides

While screening a genomic library of Acinetobacter baumannii DS002 isolated from organophosphate (OP)-polluted soils, nine ORFs were identified coding for glutathione S-transferase (GST)-like proteins. These GSTs (AbGST01-AbGST09) are phylogenetically related to a number of well-characterized GST classes found in taxonomically diverse groups of organisms. Interestingly, expression of Abgst01 (GenBank accession no. KF151191) was upregulated when the bacterium was grown in the presence of an OP insecticide, methyl parathion (MeP). The gene product, AbGST01, dealkylated MeP to desMeP. An OxyR-binding motif was identified directly upstream of Abgst01. An Abgst-lacZ gene fusion lacking the OxyR-binding site showed a drastic reduction in promoter activity. Very low β-galactosidase activity levels were observed when the Abgst-lacZ fusion was mobilized into an oxyR (GenBank accession no. KF151190) null mutant of A. baumannii DS002, confirming the important role of OxyR. The OxyR-binding sites are not found upstream of other Abgst (Abgst02-Abgst09) genes. However, they contained consensus sequence motifs that can serve as possible target sites for certain well-characterized transcription factors. In support of this observation, the Abgst genes responded differentially to different oxidative stress inducers. The Abgst genes identified in A. baumannii DS002 are found to be conserved highly among all known genome sequences of A. baumannii strains. The versatile ecological adaptability of A. baumannii strains is apparent if sequence conservation is seen together with their involvement in detoxification processes.

Changes in endolysosomal enzyme activities in cerebrospinal fluid of patients with Parkinson's disease

Parkinson's disease (PD) is characterized neuropathologically by the cytoplasmic accumulation of misfolded α-synuclein in specific brain regions. The endolysosomal pathway appears to be involved in α-synuclein degradation and, thus, may be relevant to PD pathogenesis. This assumption is further strengthened by the association between PD and mutations in the gene encoding for the lysosomal hydrolase glucocerebrosidase. The objective of the present study was to determine whether endolysosomal enzyme activities in cerebrospinal fluid (CSF) differ between PD patients and healthy controls. Activity levels of 6 lysosomal enzymes (β-hexosaminidase, α-fucosidase, β-mannosidase, β-galactosidase, β-glucocerebrosidase, and cathepsin D) and 1 endosomal enzyme (cathepsin E) were measured in CSF from 58 patients with PD (Hoehn and Yahr stages 1-3) and 52 age-matched healthy controls. Enzyme activity levels were normalized against total protein levels. Normalized cathepsin E and β-galactosidase activity levels were significantly higher in PD patients compared with controls, whereas normalized α-fucosidase activity was reduced. Other endolysosomal enzyme activity levels, including β-glucocerebrosidase activity, did not differ significantly between PD patients and controls. A combination of normalized α-fucosidase and β-galactosidase discriminated best between PD patients and controls with sensitivity and specificity values of 63%. In conclusion, the activity of a number of endolysosomal enzymes is changed in CSF from PD patients compared with healthy controls, supporting the alleged role of the endolysosomal pathway in PD pathogenesis. The usefulness of CSF endolysosomal enzyme activity levels as PD biomarkers, either alone or in combination with other markers, remains to be established in future studies.

Detection, Characterization, and Biological Effect of Quorum-Sensing Signaling Molecules in Peanut-Nodulating Bradyrhizobia

Bacteria of the genus Bradyrhizobium are able to establish a symbiotic relationship with peanut (Arachis hypogaea) root cells and to fix atmospheric nitrogen by converting it to nitrogenous compounds. Quorum sensing (QS) is a cell-cell communication mechanism employed by a variety of bacterial species to coordinate behavior at a community level through regulation of gene expression. The QS process depends on bacterial production of various signaling molecules, among which the N-acylhomoserine lactones (AHLs) are most commonly used by Gram-negative bacteria. Some previous reports have shown the production of QS signaling molecules by various rhizobia, but little is known regarding mechanisms of communication among peanut-nodulating strains. The aims of this study were to identify and characterize QS signals produced by peanut-nodulating bradyrhizobial strains and to evaluate their effects on processes related to cell interaction. Detection of AHLs in 53 rhizobial strains was performed using the biosensor strains Agrobacterium tumefaciens NTL4 (pZLR4) and Chromobacterium violaceum CV026 for AHLs with long and short acyl chains, respectively. None of the strains screened were found to produce AHLs with short acyl chains, but 14 strains produced AHLs with long acyl chains. These 14 AHL-producing strains were further studied by quantification of β-galactosidase activity levels (AHL-like inducer activity) in NTL4 (pZLR4). Strains displaying moderate to high levels of AHL-like inducer activity were subjected to chemical identification of signaling molecules by high-performance liquid chromatography coupled to mass spectrometry (LC-MS/MS). For each AHL-producing strain, we found at least four different AHLs, corresponding to N-hexanoyl-dl-homoserine lactone (C6), N-(3-oxodecanoyl)-l-homoserine lactone (3OC10), N-(3-oxododecanoyl)-l-homoserine lactone (3OC12), and N-(3-oxotetradecanoyl)-l-homoserine lactone (3OC14). Biological roles of 3OC10, 3OC12, and 3OC14 AHLs were evaluated in both AHL-producing and -non-producing peanut-nodulating strains. Bacterial processes related to survival and nodulation, including motility, biofilm formation, and cell aggregation, were affected or modified by the exogenous addition of increasing concentrations of synthetic AHLs. Our results clearly demonstrate the existence of cell communication mechanisms among bradyrhizobial strains symbiotic of peanut. AHLs with long acyl chains appear to be signaling molecules regulating important QS physiological processes in these bacteria.

Salivary β-galactosidase activity affects physiological oral malodour

ObjectivesPrevious reports have associated salivary β-galactosidase activity with non-periodontopathic oral malodour. In this study, we investigated the localization of β-galactosidase and elucidated the relationship between its enzymatic activity and physiological oral malodour. Study designFifty-six patients complaining of halitosis were separated into two groups: periodontally healthy and periodontitis. Saliva samples from the subjects were separated by centrifugation, and the level of β-galactosidase activity was measured in the supernatant, pellet lysate, and whole saliva using the chromogenic substrate o-nitrophenyl-β-d-galactopyranoside. The correlation of salivary β-galactosidase activity with breath odour and associated parameters was examined. ResultsSimilar levels of β-galactosidase activity were detected in the pellet lysate and whole saliva, but not in the saliva supernatant. Positive correlations were observed between the β-galactosidase activity in whole saliva and oral malodour levels in the periodontally healthy group, but not in the periodontitis group. In addition, the plaque index and tongue coating score were positively correlated with β-galactosidase activity in the periodontally healthy group. Overall, stimulated salivary flow and salivary pH were negatively correlated with enzyme activity. The amounts of total bacteria, Fusobacterium nucleatum, and Streptococcus salivarius were positively associated with β-galactosidase activity in the periodontally healthy group. Furthermore, the amounts of total bacteria and S. salivarius were positively associated with the amount of volatile sulphur compounds. ConclusionsOur results indicate that β-galactosidase is located on the cell surface of oral bacteria derived from dental plaque and tongue coating, and it plays an important role in producing the malodour underlying physiological oral malodour.

Production of a heterologous recombinant protein using fragments of the glyceraldehyde-3-phosphate dehydrogenase promoter from Penicillium camemberti

The biotechnological applications of cheese-ripening fungi have been limited by a lack of genetics tools, in particular the identification and characterization of suitable promoters for protein expression. In this study, the suitability of the glyceraldehyde-3-phosphate dehydrogenase (gpdP) promoter from Penicillium camemberti to drive the production of a recombinant protein was evaluated. The gpdP gene and its promoter were isolated using PCR and Genome Walker. The promoter of gpdP has two regions with high identity to the regulatory elements gpd-box and ct-box previously described in Aspergillusnidulans. Two fragments of the promoter containing the gpd- and ct-box or the ct-box alone were used to drive the in vivo production of recombinant β-galactosidase using A. nidulans as host. Our results indicate that larger fragment containing gpd-box enhances the production of β-galactosidase activity levels respect to ct-box alone, and that both boxes are necessary to obtain maximal enzymatic activity production. The smaller fragment (187 nt) containing the ct-box alone was able to trigger up to 27% of β-galactosidase activity, and to our knowledge this is the smallest fragment from a gpd gene used to produce a recombinant protein. Differences were not observed when glycerol, galactose or glucose were used as carbon sources, suggesting that the promoter activity is carbohydrate-independent. This is the first report in which a Penicillium gpd promoter is used for recombinant protein production. Our results open the way for the future development of a system for recombinant proteins expression in the biotechnologically important cheese-ripening fungus P. camemberti.

Leukotriene B4 BLT Receptor Signaling Regulates the Level and Stability of Cyclooxygenase-2 (COX-2) mRNA through Restricted Activation of Ras/Raf/ERK/p42 AUF1 Pathway

Recent studies suggest that active resolution of the inflammatory response in animal models of arthritis may involve leukotriene B(4) (LTB(4))-dependent stimulation of "intermediate" prostaglandin production, which in turn favors the synthesis of "downstream" anti-inflammatory and pro-resolving lipoxins, resolvins, and protectins. We explored a putative mechanism involving LTB(4)-dependent control of cyclooxygenase-2 (COX-2) expression, the rate-limiting step in inflammatory prostaglandin biosynthesis. Indeed, LTB(4) potently up-regulated/stabilized interleukin-1beta-induced COX-2 mRNA and protein expression under conditions of COX-2 inhibitor-dependent blockade of PGE(2) release in human synovial fibroblasts (EC(50) = 16.5 + or - 1.7 nm for mRNA; 19 + or - 2.4 nm for protein, n = 4). The latter response was pertussis toxin-sensitive, and semi-quantitative reverse transcription-PCR confirmed the quantitative predominance of the BLT2 receptor. Transfection experiments, using human COX-2 promoter plasmids and chimeric luciferase-COX-2 mRNA 3'-untranslated region (3'-UTR) reporter constructs, revealed that LTB(4) exerted its stabilizing effect at the post-transcriptional level through a 116-bp adenylate/uridylate-rich sequence in the proximal region of the COX-2 3'-UTR. Using luciferase-COX-2 mRNA 3'-UTR reporter constructs and Ras/c-Raf expression and mutant constructs, we showed that the Ras/c-Raf/MEK1/2/ERK1/2 signaling pathway mediated LTB(4)-dependent COX-2 mRNA stabilization. Knockdown experiments with specific short hairpin RNAs confirmed that LTB(4) stabilization of COX-2 mRNA was apparently mediated through the RNA-binding protein, p42 AUF1. The nuclear export of p42 AUF1 was driven by c-Raf/MEK1/2/ERK1/2 signaling and sensitive to leptomycin B treatment, suggesting a CRM1-dependent mechanism. We conclude that LTB(4) may support the resolution phase of the inflammatory response by stabilizing COX-2, ensuring a reservoir of ambient pro-resolution lipid mediators.

Use of a new catabolite repression resistant promoter isolated from Bacillus subtilis KCC103 for hyper-production of recombinant enzymes

Bacillus subtilis KCC103 hyper-produces α-amylase and the synthesis is resistant to carbon catabolite repression. The strain efficiently produced α-amylase in low cost agro-biomass based medium rich in simple sugars without catabolite repression. Here, the catabolite repression resistant promoter ( amyR4) of α-amylase was isolated from KCC103 and used to synthesize recombinant enzymes in B. subtilis. When the bgaB gene encoding β-galactosidase of Bacillus stearothermophilus was cloned and expressed under the amyR4 promoter, high level of β-galactosidase activity was found in Escherichia coli (28 U/ml)) and B. subtilis (19 U/ml). Further, the genes encoding endoxylanase ( xynA) and carboxymethyl cellulase ( bglC) from B. subtilis were cloned with signal peptides and expressed with CCR-resistant amyR4 promoter. In E. coli, the expression was intracellular with activities of cellulase and xylanase at 76 and 105 IU/ml respectively. The expression was extracellular in B. subtilis with activities at 17 and 17 IU/ml of cellulase and xylanase respectively in LB medium. When recombinant B. subtilis was cultured in LB-glucose medium, the synthesis of recombinant enzymes was not subject to catabolite repression and the expression was observed throughout the growth. This is important as glucose in the medium can prevent sporulation of the Bacillus and prevent activation of the other scavenger pathways that leads to degradation of recombinant proteins. The catabolite derepressed promoter of α-amylase from B. subtilis KCC103 can be efficiently used for overexpression of various industrial enzymes.

Herpes Simplex Virus Type 1 Thymidine Kinase Sequence Fused to the lacZ Gene Increases Levels of β-Galactosidase Activity per Genome of High-Capacity but Not First-Generation Adenoviral Vectors In Vitro and In Vivo

Increased transgene expression per vector genome is an important goal in the optimization of viral vectors for gene therapy. Herein we demonstrate that herpes simplex virus type 1 (HSV1) thymidine kinase (TK) gene sequences (1,131 bp) fused to the 3' end of lacZ increase transgene expression from high-capacity adenoviral vectors (HCAd), but not from first-generation (Ad) vectors. The woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), in contrast, increased transgene expression levels from Ad but not HCAd vectors. The differential activity of the HSV1 TK gene and WPRE sequences was detected both in vitro and in vivo and suggests potentially different mechanisms of action or the interaction of these elements with vector genomic sequences.

The Fusarium oxysporum sti35 gene functions in thiamine biosynthesis and oxidative stress response

The sti35 gene of the vascular wilt fungus Fusarium oxysporum was originally identified based on induced expression under stress conditions. In this study, the transcriptional regulation and biological function of sti35 were examined in the tomato pathogen F. oxysporum f.sp . lycopersici. Expression of sti35 was repressed by thiamine and induced by high temperatures. Sti35 transcripts were detected both during early and late stages of infection of tomato plants by F. oxysporum. Heterologous expression of the sti35 cDNA restored thiamine prototrophy in a Saccharomyces cerevisiae thi4 mutant and increased UV tolerance in a uvr − mutant of Escherichia coli. Targeted Δ sti35 knockout mutants of F. oxysporum exhibited a thiamine auxotrophic phenotype and reduced tolerance to the superoxide-generating agent menadione, indicating that Sti35 has a dual role in thiamine biosynthesis and oxidative stress response. RT-PCR analysis revealed the presence of differential RNA splicing of the second 5′-UTR intron, suggesting that thiamine may regulate sti35 expression via a post-transcriptional mechanism. F. oxysporum transformants carrying a transcriptional fusion of the sti35 promoter to the lacZ reporter gene produced high levels of β-galactosidase activity when grown in the absence, but not in the presence of thiamine. Thus, the sti35 promoter represents a useful tool for the controlled expression of genes of interest in F. oxysporum.

Aberrant Association of Promyelocytic Leukemia Protein-Retinoic Acid Receptor-α with Coactivators Contributes to Its Ability to Regulate Gene Expression

The aberrant association of promyelocytic leukemia protein-retinoic acid receptor-alpha (PML-RARalpha) with corepressor complexes is generally thought to contribute to the ability of PML-RARalpha to regulate transcription. We report here that PML-RARalpha acquires aberrant association with coactivators. We show that endogenous PML-RARalpha interacts with the histone acetyltransferases CBP, p300, and SRC-1 in a hormoneindependent manner, an association not seen for RARalpha. This hormone-independent coactivator binding activity requires an intact ligand-binding domain and the NR box of the coactivators. Confocal microscopy studies demonstrate that exogenous PML-RARalpha sequesters and colocalizes with coactivators. These observations correlate with the ability of PML-RARalpha to attenuate the transcription activation of the Notch signaling downstream effector, CBF1, and of the glucocorticoid receptor. This includes attenuation of the glucocorticoid-induced leucine zipper (GILZ) and FLJ25390 target genes of the endogenous glucocorticoid receptor. Furthermore, treatment of NB4 cells with all-trans-retinoic acid, which promotes PML-RARalpha degradation, resulted in increased activation of GILZ. On the basis of these findings, we propose a model in which the hormone-independent association between PML-RARalpha and coactivators contributes to its ability to regulate gene expression.

Pseudo-hydrodynamic Delivery of Helper-dependent Adenoviral Vectors into Non-human Primates for Liver-directed Gene Therapy

Helper-dependent adenoviral vectors (HDAds) are attractive for liver-directed gene therapy because they can mediate long-term, high-level transgene expression without chronic toxicity. However, systemic delivery requires high vector doses for efficient hepatic transduction, resulting in dose-dependent acute toxicity. Clearly, strategies to improve hepatic transduction with low vector doses are needed. In this regard, we have previously shown that hydrodynamic injection of helper-dependent adenoviral vectors into mice results in increased hepatic transduction, reduced systemic vector dissemination, and reduced pro-inflammatory cytokines compared with conventional injection and thus has the potential to improve dramatically the therapeutic index of helper-dependent adenoviral vectors. Unfortunately, the rapid, large-volume injection used in this method cannot be applied to larger animals. Therefore, we have developed a novel balloon occlusion catheter-based method to mimic hydrodynamic injection of helper-dependent adenoviral vectors into non-human primates that does not require rapid, large-volume injection. Using a low, clinically relevant vector dose, this minimally invasive method results in high-efficiency hepatic transduction with minimal toxicity and stable long-term transgene expression for at least 413 days.

A UspA2H-Negative Variant of Moraxella catarrhalis Strain O46E Has a Deletion in a Homopolymeric Nucleotide Repeat Common to uspA2H Genes

Moraxella catarrhalis strains can express either a UspA2 protein or a UspA2H protein. The latter protein is encoded by a gene that possesses a homopolymeric nucleotide tract containing eight adenine (A) residues [i.e., a poly(A) tract] which is located near the 5' end. A spontaneous UspA2H-negative variant of M. catarrhalis strain O46E, designated O46E.U2V, was found to have a uspA2H poly(A) tract that contained seven A residues. Northern blot analysis of total RNA from the O46E parent strain revealed a readily detectable uspA2H mRNA transcript, whereas little or no uspA2H transcript was detectable in total RNA from the UspA2H-negative variant O46E.U2V. The 5' end of the uspA2H genes from both the O46E parent strain and the O46E.U2V variant were ligated to a promoterless lacZ gene to prepare translational fusions for use as reporter constructs. The level of beta-galactosidase activity expressed by the fusion construct containing eight A residues in its poly(A) tract was 200-fold greater than that obtained with the construct that had seven A residues. Site-directed mutagenesis of the 5' end of the uspA2H gene confirmed that translation was initiated at a GTG codon located 21 nucleotides (nt) upstream of the poly(A) tract. Primer extension analysis determined that the transcriptional start site of the uspA2H gene was located 291 nt upstream from the GTG translational start codon. This poly(A) tract was also found to be present in the uspA2H genes of other M. catarrhalis strains.

Cytostatic Activity of Paclitaxel in Coronary Artery Smooth Muscle Cells is Mediated Through Transient Mitotic Arrest Followed by Permanent Post-Mitotic Arrest: Comparison with Cancer Cells

The anti-cancer agent paclitaxel (PTX) is an effective anti-restenosis agent on drug eluting stents, primarily due to growth inhibition of coronary artery smooth muscle cells (CASMC) across a wide dose range. In this study, we compared the effects of PTX on CASMC to apoptotic-prone HL60 leukemia cells and apoptotic-reluctant A549 lung cancer cells to assess cell survival mechanisms. In comparison to HL60 and A549 cells, CASMC had a shorter mitotic arrest and a lower mitotic index. While CASMC and A549 cells did not become apoptotic and displayed a multi-nucleated phenotype, HL60 cells showed prolonged mitotic arrest followed by apoptosis. CASMC exiting mitosis were arrested in G1 as MN tetraploid cells, with decreased levels of cyclin B1 and PCNA. CASMC remained metabolically active, becoming permanently arrested as evidenced by increased levels of β-galactosidase activity. These cells did not demonstrate elevated levels of inflammatory markers. Our findings suggest that a weak mitotic checkpoint or inhibited apoptotic cascade, or a combination of both, determine cell survival following PTX treatment. These in vitro findings suggest a mechanism for the cytostatic activity of PTX in CASMC for the inhibition of restenosis.