Increase In Beta-galactosidase Activity Research Articles

Cellular changes in boric acid-treated DU-145 prostate cancer cells

Epidemiological, animal, and cell culture studies have identified boron as a chemopreventative agent in prostate cancer. The present objective was to identify boron-induced changes in the DU-145 human prostate cancer cell line. We show that prolonged exposure to pharmacologically-relevant levels of boric acid, the naturally occurring form of boron circulating in human plasma, induces the following morphological changes in cells: increases in granularity and intracellular vesicle content, enhanced cell spreading and decreased cell volume. Documented increases in β-galactosidase activity suggest that boric acid induces conversion to a senescent-like cellular phenotype. Boric acid also causes a dose-dependent reduction in cyclins A–E, as well as MAPK proteins, suggesting their contribution to proliferative inhibition. Furthermore, treated cells display reduced adhesion, migration and invasion potential, along with F-actin changes indicative of reduced metastatic potential. Finally, the observation of media acidosis in treated cells correlated with an accumulation of lysosome-associated membrane protein type 2 (LAMP-2)-negative acidic compartments. The challenge of future studies will be to identify the underlying mechanism responsible for the observed cellular responses to this natural blood constituent.

Directed Evolution and Identification of Control Regions of ColE1 Plasmid Replication Origins Using Only Nucleotide Deletions

Genes can be mutated by altering DNA content (base changes) or DNA length (insertions or deletions). Most in vitro directed evolution processes utilize nucleotide content changes to produce DNA libraries. We tested whether gain of function mutations could be identified using a mutagenic process that produced only nucleotide deletions. Short nucleotide stretches were deleted in a plasmid encoding lacZ, and screened for increased beta-galactosidase activity. Several mutations were found in the origin of replication that quantitatively and qualitatively altered plasmid behavior in vivo. Some mutations allowed co-residence of ColE1 plasmids in Escherichia coli, and implicate hairpin structures II and III of the ColE1 RNA primer as determinants of plasmid compatibility. Thus, useful and unexpected mutations can be found from libraries containing only deletions.

Characterization of Tetracycline-Inducible BitransgenicKrt12rtTA/+/tet-O-LacZMice

To prepare binary transgenic mouse lines that overexpress reporter genes in a corneal-epithelium-specific manner when induced by doxycycline. A gene-targeting construct containing an internal ribosomal entry site-reverse tetracycline transcription activator (IRES-rtTA) cassette was inserted into the Krt12 allele (keratin 12 gene) to produce a knock-in Krt12(rtTA/+) mouse line through gene-targeting techniques. The Krt12(rtTA/+) knock-in mice were bred with tet-O-LacZ reporter mice to obtain Krt12(rtTA/+)/tet-O-LacZ bitransgenic mice. The expression of the LacZ gene was induced in bitransgenic mice by administration of doxycycline in the drinking water and chow. Administration of doxycycline induced a 15-fold increase of beta-galactosidase activity in the cornea of adult bitransgenic mice (Krt12(rtTA/+)/tet-O-lacZ). Administration of doxycycline either to single transgenic Krt12(rtTA/+) or tet-O-LacZ mice as a control did not induce overexpression of LacZ as it did in the bitransgenic mice. The induction of beta-galactosidase enzyme activity by doxycycline in bitransgenic mice took place in 24 hours and reached a plateau by 2 days. Histochemical analysis also showed that beta-galactosidase induction was limited to the corneal epithelium of bitransgenic mice fed doxycycline. The increased beta-galactosidase activity in corneal epithelium caused by doxycycline returned to basal levels in 4 weeks after the antibiotics were omitted from the diet. A binary mouse model has been successfully established that conditionally overexpresses reporter genes in corneal epithelium. This mouse model will be useful in elucidating signaling pathways of various growth factors and cytokines and gene functions in the maintenance of homeostasis and pathogenesis in the adult mouse cornea.

Activated sludge deflocculation under temperature upshifts from 30 to 45°C

Twelve independent batch experiments (<9h) with fresh municipal activated sludge were conducted to assess the occurrence and the mechanisms of deflocculation under a temperature shift from 30 to 45 degrees C. In each experiment, a transient reactor (2 L) was subjected to the temperature shift and a control reactor was operated at a constant temperature of 30 degrees C. The occurrence of deflocculation was demonstrated by the increase in turbidity and in the concentrations of biopolymers in the sludge supernatant from the transient reactor. The maximum levels of proteins in the supernatants ranged from 53 to 81 mg/L, for DNA from 34 to 36 mg/L, for humic compounds from 20 to 40 mg/L, and for carbohydrates from 21 to 31 mg/L. All the biopolymer concentrations in the control reactor remained below 5-10 mg/L. The release of biopolymers was accompanied by an increase in sludge supernatant conductivity (16-32% increase, up to 1.20 mS/cm), soluble chemical oxygen demand (from 129 to 440 mg/L), total suspended solids (>25 mg/L up to 128 mg/L), and a decrease in the mixed liquor volatile suspended solids (up to 11%). The temperature shift was also found to inhibit microbial metabolism by reducing the sludge biomass substrate removal capacity, as measured by oxygen-uptake rates. The temperature shift had a marginal effect causing sludge lysis (as an increase in beta-galactosidase activity) and had no significant impact on sludge viability (live/dead ratio of bacterial cells). It was concluded that sludge deflocculation under a temperature shift from 30 to 45 degrees C involves the solubilisation of extracellular polymeric substances from the flocs and likely also floc fragmentation. In addition, sludge deflocculation and the inhibition of microbial metabolism explain the poor treatment performance observed in previous continuous reactors under similar temperature shifts.

Modeling gene expression in the mouse uterine horn by in vivo adenovirus-mediated gene delivery

Endometrium is a unique tissue that is prepared for implantation of blastocyst during each menstrual cycle by expression of genes during a defined period of endometrial receptivity. Induction of gene over-expression in endometrium allows gaining insight on the role that genes play in endometrial function. Here, we show that induction of a state of gene over-expression in endometrium is feasible by in vivo gene delivery by transduction with adenovirus. Analysis of endometrium following adenoviral transduction of LacZ showed increased beta-galactosidase activity in endometrial glands as early as 24 hours following in vivo gene transfer. By 72 hour, the expression was uniformly strong throughout the uterine horn. Viral transduction was efficient in the range of 0.24-24x10(8) pfu (4.8-480x10(8) particles) in normal, pregnant and decidualized mouse uterine horns. These findings show that induction of a state of gene over-expression can be successfully attained in endometrium by adenoviral gene delivery.

Tumor necrosis factor alpha induces senescence and chromosomal instability in human leukemic cells.

Previous studies have documented that Tumor necrosis factor alpha (TNFalpha) is a potent negative regulator of normal hematopoiesis. However, the mechanism by which TNFalpha acts at the cellular level is not totally understood. Although apoptotic cell killing appears to be the most common cellular effect of TNFalpha, other studies suggest that this cytokine may elicit other cellular responses such as prolonged growth inhibition. In this context, we have investigated whether TNFalpha may induce senescence in hematopoietic cells, which display intrinsic defect in the apoptotic machinery. The present study described that, in the leukemic KG1 cells, TNFalpha induced no apoptosis but a senescence state characterized by prolonged growth arrest, increased beta-galactosidase activity, p21WAF-1 induction, decreased telomerase activity, telomeric disturbances (shortening, losses, fusions), and additional chromosomal aberrations. Telomerase inhibition correlated with reduced levels of hTERT transcripts. GM-CSF prevented TNFalpha effects and allowed leukemic cells to recover growth capacity. Finally, our study shows for the first time that, at least in some hematopoietic cells, TNFalpha may induce senescence with important functional consequences, including sustained growth inhibition and genetic instability, and that this cellular response is efficiently regulated by hematopoietic growth factors.

Biologic modification of ligamentum flavum cells by marker gene transfer and recombinant human bone morphogenetic protein-2.

The study involves an in vitro experiment using human ligamentum flavum (LF), adenovirus lacZ construct (Ad/lacZ), and recombinant human bone morphogenetic protein-2 (BMP-2). To demonstrate the feasibility of marker gene transfer to human LF cells and the effect of BMP-2 on the osteogenic differentiation of human LF cells. BMP-2 is a widely known pivotal osteoinductive agent. Clinically and experimentally, BMP-2 has proven to be an effective in spinal fusion. Degenerated LF has only been implicated to be of pathophysiological significance in spinal stenosis. However, biologic modifications of LF to enhance osteogenesis have not been attempted previously. Human LF and cancellous bone from the ilium were harvested from patients with lumbar spinal stenosis. LF cells and osteoblasts were isolated and cultured, and adenovirus lacZ construct (Ad/ lacZ), luciferase construct (Ad/luciferase), and BMP-2 were designed and produced. LF cell cultures were then exposed to various concentrations of Ad/lacZ (25, 50, 75, 100, 150 multiplicity of infection) and BMP-2 (50, 100, 500, 1,000, and 1,500 ng/mL). Osteoblast cultures were used as a positive control for LF culture. LF cell cultures with Ad/luciferase served as viral controls for culture with Ad/ lacZ. The transgene expression of lacZ was assessed by X-gal stain and beta-galactosidase assay. Alkaline phosphatase, Von Kossa, and Alizarin red-S stains were used to confirm osteogenic differentiation and bone nodule formation. Immunocytochemical staining was also performed to detect osteocalcin expression. LF cell cultures transduced with Ad/lacZ showed extensive X-gal expression and increased beta-galactosidase activity compared to viral (Ad/luciferase) and saline controls. In LF cultures treated with BMP-2, robust alkaline phosphatase expression, and bone nodule formations were observed as evidenced by positive Von Kossa and Alizarin red-S staining, and the strong expression of osteocalcin. The osteogenic response of LF cells to BMP-2 was dose dependent. Human LF cells were found to be susceptible to adenovirus-mediated marker gene transfer, which offers the possibility of a new range of possible genetic modifications. In human LF cells, BMP-2 was found to markedly up-regulate the expression of osteogenic phenotypes and to induce bone nodule formation. The results of this study support the notion that biologically modified LF cells, i.e., LF cells treated with BMP-2, or with adenovirus-mediated BMP-2 cDNA gene transfer, may facilitate spinal fusion.

Environmental factors affecting transcription of the human L1 retrotransposon. II. Stressors.

Retrotransposons have clearly molded the structure of the human genome. The reverse transcriptase coded for by long interspersed nuclear elements (LINEs) accounts for 35% of the human genome, with 8-9 x 10(5) copies of the most common human LINE element, L1Hs. Retrotransposons cycle through an RNA intermediate with transcription as the rate limiting step. Because various retrotransposons have been demonstrated to be induced by environmental stimuli, we investigated the response of the L1Hs promoter to various agents. L1Hs promoter activity was analyzed by transfecting an L1Hs-expressing cell line with plasmids containing one of two L1Hs promoters fused to the LacZ reporter gene. L1Hs promoter activity was then monitored with a beta-galactosidase assay. Treatment with UV light and heat shock resulted in a small increase in beta-galactosidase activity from one promoter, while treatment with tetradecanoylphorbol 13-acetate resulted in small increases in beta-galactosidase activity from both promoters. No increase in beta-galactosidase activity was observed after exposure to X-rays or hydrogen peroxide.

Beta-Galactosidase activity of Escherichia coli under long-term starvation, alterations in temperature, and different nutrient conditions in lake water.

beta-Galactosidase activity of Escherichia coli was investigated in response to long-term starvation, changes in temperature and the presence of certain nutrient sources in lake water. beta-Galactosidase activity decreased markedly in filtered-autoclaved lake water at 25 degrees C and 37 degrees C, whereas it remained almost constant at 4 degrees C and 15 degrees C for 60 days. Increases in beta-galactosidase activity were observed in response to the following nutrient sources: glycine, serine, methionine and ammonium sulfate at 4 degrees C; glycine and ammonium sulfate at 15 degrees C; glycine, serine, methionine and ammonium sulfate at 30 degrees C. Glycine addition led to an increase in beta-galactosidase activity of almost five and seven orders of magnitude at 15 degrees C and 30 degrees C, respectively. In addition, L-methionine had the strongest influence on beta-galactosidase activity, which was detected as an increase of seven and eleven orders of magnitude at 4 degrees C and 30 degrees C, respectively. The effect of several amino acids and other nitrogen sources depended on the concentration of the nutrient source and the temperature. The results showed that, in lake water, long-term starvation, temperature change, and variations in nitrogen sources alter beta-galactosidase activity. Those effects should be taken into account when monitoring coliforms from the environment.

Expression of caveolin-1 induces premature cellular senescence in primary cultures of murine fibroblasts.

Caveolae are vesicular invaginations of the plasma membrane. Caveolin-1 is the principal structural component of caveolae in vivo. Several lines of evidence are consistent with the idea that caveolin-1 functions as a "transformation suppressor" protein. In fact, caveolin-1 mRNA and protein expression are lost or reduced during cell transformation by activated oncogenes. Interestingly, the human caveolin-1 gene is localized to a suspected tumor suppressor locus (7q31.1). We have previously demonstrated that overexpression of caveolin-1 arrests mouse embryonic fibroblasts in the G(0)/G(1) phase of the cell cycle through activation of a p53/p21-dependent pathway, indicating a role of caveolin-1 in mediating growth arrest. However, it remains unknown whether overexpression of caveolin-1 promotes cellular senescence in vivo. Here, we demonstrate that mouse embryonic fibroblasts transgenically overexpressing caveolin-1 show: 1) a reduced proliferative lifespan; 2) senescence-like cell morphology; and 3) a senescence-associated increase in beta-galactosidase activity. These results indicate for the first time that the expression of caveolin-1 in vivo is sufficient to promote and maintain the senescent phenotype. Subcytotoxic oxidative stress is known to induce premature senescence in diploid fibroblasts. Interestingly, we show that subcytotoxic level of hydrogen peroxide induces premature senescence in NIH 3T3 cells and increases endogenous caveolin-1 expression. Importantly, quercetin and vitamin E, two antioxidant agents, successfully prevent the premature senescent phenotype and the up-regulation of caveolin-1 induced by hydrogen peroxide. Also, we demonstrate that hydrogen peroxide alone, but not in combination with quercetin, stimulates the caveolin-1 promoter activity. Interestingly, premature senescence induced by hydrogen peroxide is greatly reduced in NIH 3T3 cells harboring antisense caveolin-1. Importantly, induction of premature senescence is recovered when caveolin-1 levels are restored. Taken together, these results clearly indicate a central role for caveolin-1 in promoting cellular senescence and they suggest the hypothesis that premature senescence may represent a tumor suppressor function mediated by caveolin-1 in vivo.

Environmental factors affecting transcription of the human L1 retrotransposon. I. Steroid hormone-like agents.

The L1 retrotransposon has significantly shaped the structure of the human genome. At least 30% of human genome sequence can be attributed to L1 reverse transcriptase activity. There are 10(5) copies of the human L1 retrotransposon, L1Hs, most of which are defective, although approximately 8-9 x 10(3) are full length. L1Hs elements transpose through an RNA intermediate and transcription is thought to be the rate limiting step in retrotransposition. Because transcription of retrotransposons in a variety of organisms has been shown to respond to environmental stimuli, we investigated the influence of various agents on transcription from two different L1Hs promoters. The activity of the L1Hs promoters was analyzed by transfecting L1Hs-expressing cell lines with plasmids containing the L1Hs promoters fused to the LacZ reporter gene and monitoring expression with a beta-galactosidase assay. Small increases in beta-galactosidase activity were observed with both L1Hs promoters after treatment with serum, testosterone, dihydrotestosterone and organochloride pesticides, indicating that these agents can influence L1Hs transcription.

Expression, autoregulation, and DNA binding properties of the Mycobacterium tuberculosis TrcR response regulator.

The TrcRS two-component system of Mycobacterium tuberculosis is comprised of the TrcS histidine kinase and the TrcR response regulator, which is homologous to the OmpR class of DNA binding response regulators. Reverse transcription-PCRs with total RNA showed that the trcR and trcS two-component system genes are transcribed in broth-grown M. tuberculosis. Analysis of the trcR and trcS genes using various SCOTS (selective capture of transcribed sequences) probes also confirmed that these genes are expressed in broth-grown cultures and after 18 h of M. tuberculosis growth in cultured human primary macrophages. To determine if the TrcR response regulator is autoregulated, a trcR-lacZ fusion plasmid and a TrcR expression plasmid were cotransformed into Escherichia coli. Upon induction of the TrcR protein, there was a >500-fold increase in beta-galactosidase activity from the trcR-lacZ fusion, indicating that TrcR is involved in transcriptional autoactivation. Gel mobility shift assays with the trcR promoter and TrcR established that the response regulator was autoregulating via direct binding. By use of a delimiting series of overlapping trcR PCR fragments in gel mobility shift assays with TrcR, an AT-rich region of the trcR promoter was shown to be essential for TrcR binding. Additionally, this AT-rich sequence was protected by TrcR in DNase I protection assays. To further analyze the role of the AT-rich region in TrcR autoregulation, the trcR promoter was mutated and analyzed in lacZ transcriptional fusions in the presence of TrcR. Alteration of the AT-rich sequence in the trcR promoter resulted in the loss of trcR transcriptional activation in the presence of TrcR. This report indicates that the M. tuberculosis TrcR response regulator activates its own expression by interacting with the AT-rich sequence of the trcR promoter.

Identification and role in virulence of putative iron acquisition genes from Corynebacterium pseudotuberculosis.

Four genes, fagA, B, C and D, encoding products with 32-47% identity to proteins involved in bacterial iron uptake systems, were identified immediately downstream of the Corynebacterium pseudotuberculosis phospholipase D gene. beta-Galactosidase assays on a C. pseudotuberculosis strain carrying a fagA-lacZ fusion indicated that the putative fagABC operon was poorly expressed in iron-rich media. However, similar experiments in iron-limited media resulted in an approximately three-fold increase in beta-galactosidase activity, suggesting that this operon is regulated by iron in vitro. Although no defect in iron utilization could be determined for a C. pseudotuberculosis fagB(C) mutant in vitro, this mutant showed reduced virulence compared to wild-type in a goat model of caseous lymphadenitis. Thus, expression of the fag genes in the host appears to contribute to virulence.

Genotoxicity of 5-aminolevulinic and 4,5-dioxovaleric acids in the salmonella/microsuspension mutagenicity assay and SOS chromotest.

5-Aminolevulinic acid (ALA) is a heme precursor that accumulates in some porphyric disorders and in lead poisoning which can undergo metal-catalyzed oxidation producing reactive oxygen species and the keto-aldehyde, 4,5-dioxovaleric acid (DOVA). Evidence in vitro of ALA-induced DNA lesions suggests that ALA and DOVA have mutagenic potential that could possibly contribute to an increased frequency of hepatocellular carcinoma (HCC) in patients with acute intermittent porphyria (AIP). In this study, we evaluated the genotoxic potential of ALA and DOVA. In the absence of exogenous metabolic activation, ALA and DOVA were mutagenic in Salmonella typhimurium tester strain TA104. ALA was also mutagenic in S. typhimurium TA102, but not in TA98, TA100, or TA1535, indicating an oxidative mechanism. Removal of H(2)O(2) with catalase gave only partial protection, suggesting generation of other mutagenic species. Both ALA and DOVA damaged the DNA of Escherichia coli PQ37, inducing the SOS response detected by an increase in beta-galactosidase activity. These results verified the potential mutagenic activity of ALA and DOVA and reinforce the hypothesis that DNA damage induced by ALA may be associated with the development of HCC in individuals suffering from AIP.

Expression patterns of cell wall-modifying enzymes during grape berry development.

During ripening of grape (Vitis vinifera L.) berries, softening occurs concomitantly with the second growth phase of the fruit and involves significant changes in the properties of cell wall polysaccharides. Here, the activities of enzymes that might participate in cell wall modification have been monitored throughout berry development. Alpha-galactosidase (EC 3.2.1.22), beta-galactosidase (EC 3.2.1.23) and pectin methylesterase (EC 3.1.1.11) activities were present, but no polygalacturonase (EC 3.2.1.15), cellulase (EC 3.2.1.4), xyloglucanase (xyloglucan-specific cellulase EC 3.2.1.4) or galactanase (EC 3.2.1.89) could be detected. The accumulation of mRNAs encoding wall-modifying enzymes was examined by northern hybridization analysis. Transcripts for beta-galactosidase, pectin methylesterase, polygalacturonase, pectate lyase (EC 4.2.2.2) and xyloglucan endotransglycosylase (EC 2.4.1.207) were present during ripening, although polygalacturonase activity had not been detected in berry extracts. Cellulases could not be detected in ripening berries, either at the enzyme or mRNA levels. The increase in beta-galactosidase activity and mRNA is consistent with the observed decrease in type-I arabinogalactan content of the walls during ripening, and the detection of polygalacturonase and pectate lyase mRNAs might explain the increased solubility of galacturonan in walls of ripening grapes. Thus, the modification of cell wall polysaccharides during softening of grape berries is a complex process involving subtle changes to different components of the wall, and in many cases only small amounts of enzyme activity are required to effect these changes.

The Osteoblast-specific Transcription Factor Cbfa1 Contributes to the Expression of Osteoprotegerin, a Potent Inhibitor of Osteoclast Differentiation and Function

Bone formation and resorption are tightly coupled under normal conditions, and the interaction of osteoclast precursors with cells of the osteoblast lineage is a prerequisite for osteoclast formation. Cbfa1 is an osteoblast-specific transcription factor that is essential for osteoblast differentiation and bone formation. At present, it is not known whether Cbfa1 regulates any of the osteoblast-derived factors involved in the bone resorption pathway. Osteoprotegerin (OPG) is an osteoblast-secreted glycoprotein that functions as a potent inhibitor of osteoclast differentiation and bone resorption. Cloning and computer analysis of a 5.9-kilobase human OPG promoter sequence revealed the presence of 12 putative Cbfa1 binding elements (osteoblast-specific element 2 (OSE(2))), suggesting a possible regulation of OPG by Cbfa1. We cloned the promoter upstream of the beta-galactosidase reporter gene (pOPG5. 9betagal) and evaluated whether Cbfa1 could regulate its expression in transient transfection assays. The 5.9-kilobase promoter directed increased levels of reporter gene expression, reminiscent of OPG protein levels in osteoblastic cell lines (BALC and U2OS) as compared with the nonosteoblastic cell line COS1. Cotransfection of a Cbfa1 expression construct along with pOPG5.9betagal reporter construct led to 39-, 7-, and 16-fold increases in beta-galactosidase activity in COS1, BALC, and U2OS cells, respectively. Removal of all the putative OSE(2) elements led to an almost complete loss of transactivation. Mutational analysis demonstrated that the proximal OSE(2) element contributes to a majority of the effects of Cbfa1, and Cbfa1 bound to the proximal element in a sequence-specific manner. Further, overexpression of Cbfa1 led to a 54% increase in OPG protein levels in U2OS cells. These results indicate that Cbfa1 regulates the expression of OPG, thereby further contributing to a molecular link between bone formation and resorption.

Involvement of mitochondrial aldehyde dehydrogenase ALD5 in maintenance of the mitochondrial electron transport chain inSaccharomyces cerevisiae

The physiological role of mitochondrial aldehyde dehydrogenase (ALD5) was investigated by analysis of the ald5 mutant (AKD321) in Saccharomyces cerevisiae. K(+)-activated ALDH activity of the ald5 mutant was about 80% of the wild-type in the mitochondrial fraction, while the respiratory activity of the ald5 mutant was greatly reduced. Cytochrome content was also reduced in the ald5 mutant. Enzymatic analysis revealed that the alcohol dehydrogenase activity of the ald5 mutant was higher than that of the wild-type, while glycerol 3-phosphate dehydrogenase activity was the same in the two strains. Ethanol as a carbon source or addition of 1 M NaCl with glucose as the carbon source in the growth medium increased beta-galactosidase activity from an ALD5-lacZ fusion. Overexpression of another mitochondrial ALDH gene (ALD7) had no effect on increasing respiratory function of the ald5 mutant, but showed improved growth on ethanol. These observations show that mitochondrial ALD5 plays a role in regulation or biosynthesis of electron transport chain components.

Caenorhabditis elegans as a biomonitor for immunological stress in nematodes.

An experimental system has been developed using Caenorhabditis elegans (Secernentea: Rhabditida), to monitor immunological stress in nematodes. The transgenic C. elegans strain PC72 carries a lacZ reporter gene fused to a C. elegans hsp16-1 gene, which is inducible for beta-galactosidase activity at the heat stress temperature of 26 degrees C. The investigate the possibility of using PC72 to monitor immunological stress, its surface coat was targeted, to mimic immune attack, by raising immune sera against surface coat components selectively removed by the cationic detergent cetyltrimethylammoniunm bromide. Initially, a highly significant induction of beta-galactosidase activity was seen in PC72 incubated in either surface-reactive or naïve rabbit serum. Complement (C3) was detected over the entire surface of adult PC72 and was thought to be responsible for stress-induction with naïve sera. When the immunoglobulin (Ig)G fraction of naïve sera was used in isolation, no stress-induction was seen. In contrast, a two-fold increase in beta-galactosidase activity was seen in the presence of surface-reactive IgG (SR-IgG) which recognised surface components of between 6 and 40 kDa in western blot. The belief that surface reactive IgG could induce a stress response was reinforced by analysis of hsp-16 protein expression. Cationised ferritin was then used to assess whether stress-induction was truly a surface reactive event; binding of cationised ferritin to the nematode surface also resulted in two-fold induction of beta-galactosidase activity. To investigate the downstream biological effects of stress induction, worm growth and fecundity were measured in the presence of IgG preparations. A significant reduction was seen in both worm length and fecundity only when larvae were incubated in surface-reactive IgG, compared to both naïve IgG and K-medium controls. In conclusion, it would appear that C. elegans is a suitable model to monitor the induction of immunological stress at the level of the nematode surface coat. Given the ability of nematode surface antigens to protect the vaccinated host in animal model systems, and the close phylogenetic relationships which exist between C. elegans and nematodes of medical and veterinary importance, it is conceivable that the immunological targets in or on the surface of C. elegans warrant rapid identification.

Transgene expression in malignant glioma using a replication-defective adenoviral vector containing the Egr-1 promoter: activation by ionizing radiation or uptake of radioactive iododeoxyuridine.

One approach to improving the specificity of gene therapy involves using radiosensitive promoters to activate gene expression selectively in the radiation field. In this study, we evaluated the ability of irradiation to regulate the transcription of a recombinant replication-defective adenovirus vector, Ad.Egr-1/lacZ, containing the radiation-inducible Egr-1 promoter driving the beta-galactosidase reporter gene in glioma cells. Transcripts of the Egr-1 gene in human and rat glioma cells were induced following irradiation with as little as 2 Gy. This dose was 10-fold less than previously reported, and comparable to doses of irradiation used clinically in standard fractionated radiotherapy for brain tumors. When 9L rat gliosarcoma cells were infected with Ad.Egr-1/lacZ in vitro and exposed to 2 Gy of external beam irradiation, there was a threefold increase in beta-galactosidase expression. Irradiation of intracerebral 9L tumors infected with the Ad.Egr-1/lacZ virus, using either external beam radiotherapy (2 Gy) or the thymidine analog 5-iodo-2'-deoxyuridine radiolabeled with the Auger electron emitter iodine-125 ([125I]IdUrd), also resulted in increased beta-galactosidase activity of the tumor cells. These results indicate that the use of viral vectors containing radiation-inducible promoters represents a novel therapeutic approach that enables gene therapy to be spatially and temporally regulated by ionizing radiation. These findings also support a potential role for radiation-inducible promoters in the treatment of malignant brain tumors.

The ZnuABC high-affinity zinc uptake system and its regulator Zur in Escherichia coli.

In Escherichia coli, lacZ operon fusions were isolated that were derepressed under iron repletion and repressed under iron depletion. Two fusions were localized in genes that formed an operon whose gene products had characteristics of a binding protein-dependent transport system. The growth defect of these mutants on TY medium containing 5mM EGTA was compensated for by the addition of Zn2+. In the presence of 0.5mM EGTA, only the parental strain was able to take up 65Zn2+. This high-affinity transport was energized by ATP. The genes were named znuACB (for zinc uptake; former name yebLMI) and localized at 42 min on the genetic map of E. coli. At high Zn2+ concentrations, the znu mutants took up more 65Zn2+ than the parental strain. The high-affinity 65Zn2+ uptake was repressed by growth in the presence of 10 microM Zn2+. A znuA-lacZ operon fusion was repressed by 5 microM Zn2+ and showed a more than 20-fold increase in beta-galactosidase activity when Zn2+ was bound to 1.5 microM TPEN [tetrakis-(2-pyridylmethyl) ethylenediamine]. To identify the Zn2+-dependent regulator, constitutive mutants were isolated and tested for complementation by a gene bank of E. coli. A complementing gene, yjbK of the E. coli genome, was identified and named zur (for zinc uptake regulation). The Zur protein showed 27% sequence identity with the iron regulator Fur. High-affinity 65Zn2+ transport of the constitutive zur mutant was 10-fold higher than that of the uninduced parental strain. An in vivo titration assay suggested that Zur binds to the bidirectional promoter region of znuA and znuCB.