Β-galactosidase Activity Assay Research Articles

Efficient gene transfer into the epithelial cell layer of embryonic mouse intestine using low-voltage electroporation

Background & Aims : Gene-targeted and transgenic mice have provided profound insights into the genetic control of intestinal development and differentiation. Here we describe a complementary approach using an innovative embryonic gut culture system in which the tubular architecture of the gut is maintained and rapid analysis of gene function can be achieved by introduction of expression constructs into the epithelial cell layer by using low-voltage square-wave electroporation. Methods : Electroporation of embryonic gut explants was optimized by using an electrosquareporator to vary the voltage and number of electric pulses. The pulses were applied unidirectionally to confine electroporation of plasmids to one side of the epithelium, providing experimental and control regions within the same gut segment. The expression of constructs was examined by fluorescence and confocal microscopy, immunohistochemistry, and β-galactosidase activity assays. Results : Conditions were identified in which a high proportion of cells (5% to 20%) within the epithelial cell layer of gut explants expressed transfected genes without cell death. Electroporation of a construct encoding a Cre-enhanced green fluorescent protein (EGFP) fusion protein into explants from ROSA26 reporter animals resulted in extensive activation of β-galactosidase expression, indicating that the electroporated construct produced functionally active protein. Conclusions : A novel embryonic gut culture system has been developed to explore the influence of genes on intestinal epithelial cell behavior in situ. Complementing the powerful genetics available in the mouse, electroporation provides a rapid method for the analysis of genes that play pivotal roles in intestinal differentiation.

Mechanisms of microwave irradiation involved in the destruction of fecal coliforms from biosolids

Microwaves have been found to be effective in destructing pathogens in sewage sludge (biosolids) (75th Annual Water Environment Federation Conference, Chicago, September 29–October 2, 2002; Third World Water Congress, International Water Association, Melbourne, Australia, April 7–12, 2002). Mechanisms and roles of microwaves on fecal coliform destruction were investigated using bacterial viability tests, electron transport system (ETS) and β-galactosidase activity assays, gel electrophoresis, and genomic deoxyribonucleic acid (DNA) optical density (OD) measurements with fecal coliforms isolated from biosolids. Bacterial viability tests demonstrated cell membrane damage as microwave irradiation intensity and temperature increased. Above 60±3°C, viable cells were rarely found when pure fecal coliforms were irradiated with microwaves. ETS and β-galactosidase activity assays revealed increased activity for externally heated samples due to fecal coliform growth but decreased activity for microwave-irradiated samples as temperature was increased from 20°C to 57°C, indicating other destruction mechanisms besides heating. Between 57°C and 68°C, microwave irradiation led to a more rapid decrease in activity than external heating by convection. Above 68°C, bacterial activity almost ceased for both pretreatments. DNA bands in gel electrophoresis tests and OD of genomic DNA decreased more rapidly for microwave-irradiated samples than for externally heated samples, implying that microwaves disrupted DNA in fecal coliform cells at lower temperatures than external heating. Microwave irradiation of sludge appears to be a viable and economical method of destructing pathogens and generating environmentally safe sludge.

Receptor-independent augmentation of adenovirus-mediated gene transfer with chitosan in vitro

Recombinant adenovirus is one of the most widely used viral vectors for gene delivery. This study was designed to evaluate the ability of chitosan, a cationic, linear polysaccharide composed of β(1,4) linked glucosamine partly containing N-acetyl-glucosamine, to enhance the in vitro infectivity of adenovirus to mammalian cells. Wild type and a fiber-mutant replication-defective recombinant adenoviruses expressing β-galactosidase were used. In the latter, an RGD peptide, the binding site for α v β 3 and α v β 5 integrin, was introduced in the fiber knob enabling adenovirus receptor-independent viral infection. Enhanced effect of chitosan on the infectivity of both adenoviruses was observed in Chinese hamster ovary cells that do not express the receptor for adenovirus with β-galactosidase activity assay and x-gal staining. These data indicate the receptor-independent mechanism(s) for this enhancement effect. In addition, we found that pH of the culture medium, and molecular mass and concentration of chitosan are also critical factors. Thus, the highest effect was obtained with 0.1–1 μg/ml of chitosan with molecular mass of 19K and 40K in the culture medium of pH 6.4; on the other hand, the effect was negligible with the higher chitosan concentrations (10 μg/ml or more), lower or higher molecular mass (11K and 110K) of chitosan, or at pH of 7.4. Studies using several cell lines with variable levels of adenoviral infectivity revealed that this enhanced effect is evident in the cells with poor infectivity to adenovirus. Since chitosan is biocompatible and inexpensive, these data indicate that chitosan may be a potential candidate for a non-viral vector to safely increase adenoviral infectivity to mammalian cells, particularly those with poor susceptibility to adenoviral infection.

Measuring β-Galactosidase Activity in Bacteria: Cell Growth, Permeabilization, and Enzyme Assays in 96-Well Arrays

We describe a high-throughput procedure for measuring β-galactosidase activity in bacteria. This procedure is unique because all manipulations, including bacterial growth and cell permeabilization, are performed in a 96-well format. Cells are permeabilized by chloroform/SDS treatment directly in the 96-well blocks and then transferred to 96-well microplates for standard colorimetric assay of β-galactosidase activity as described by Miller [J. H. Miller (1972) Experiments in Molecular Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY]. Absorbance data are collected with a microplate reader and analyzed using a Microsoft Excel spreadsheet. The β-galactosidase specific activity values obtained with the high-throughput procedure are identical to those obtained by the traditional single-tube method of Miller. Thus, values obtained with this procedure may be expressed as Miller units and compared directly to Miller units reported in the literature. The 96-well format for permeabilization and assay of enzyme specific activity together with the use of 12-channel and repeater pipettors enables efficient processing of hundreds of samples in an 8-h day.

Bradyrhizobium japonicum mutants with enhanced sensitivity to genistein resulting in altered nod gene regulation.

Bradyrhizobium japonicum mutants with altered nod gene induction characteristics were isolated by screening mutants for genistein-independent nod gene expression. Plasmid pZB32, carrying a nodY::lacZ transcriptional gene fusion, was introduced into B. japonicum cells that had been subjected to UV mutagenesis. Ten independent transformants producing a blue color on plates containing 5bromo-4chloro-3indolyl-beta-D-galactopyranoside but lacking genistein, indicative of constitutive expression of the nodY::lacZ reporter gene, were isolated. Beta-galactosidase activity assays revealed that while all of the 10 strains were sensitive to low concentrations of genistein, none exhibited truly constitutive nodY::lacZ expression in liquid culture. Soybean plants inoculated with three of the mutants were chlorotic and stunted, with shoot dry weights close to those of the uninoculated plants, indicating the absence of nitrogen fixation. Differences in the kinetics of nodY::lacZ expression and lipochitin oligosaccharide Nod signal production suggested that the strains carried different mutations. Some of these strains may be useful in mitigating the low root zone temperature-associated delay in soybean nodulation at the northern extent of soybean cultivation.

Application of Hydrostatic Pressure Facilitates ex Vivo Adenovirus Gene Transfer into Rabbit Aortas

We investigated whether application of hydrostatic pressure facilitates ex vivo adenovirus gene transfer into rabbit aortas. Assay of β-galactosidase activity was performed in the aortas subjected to adenovirus encoding LacZ gene (Ad-LacZ) transfer. Application of hydrostatic pressure at 8 atmospheres during Ad-LacZ gene transfer (1010 pfu/mL, 10 min) to aortic segments resulted in an approximately 4.5-fold increase in transgene efficiency. X-Gal staining showed predominant β-galactosidase activity in the endothelial and the adventitial cells in the aorta subjected to pressure-supported Ad-LacZ gene transfer. Then we examined the effect of heme oxygenase-1 (HO-1), which catabolizes heme to carbon monoxide and biliverdin, by transferring adenovirus encoding HO-1 (Ad-HO-1) into rabbit aortas using this pressurization system. The Ad-HO-1-infected aortic segment showed a significantly decreased contractile response to phenylephrine compared to the Ad-LacZ-infected aortic segment. Pressure-supported adenovirus gene transfer may increase the feasibility of exploiting intraoperative adenovirus-mediated gene transfer.

Fluorogenic Substrates Based on Fluorinated Umbelliferones for Continuous Assays of Phosphatases and β-Galactosidases

Fluorogenic substrates based on 4-methylumbelliferone (4-MU) have been widely used for the detection of phosphatase and glycosidase activities. One disadvantage of these substrates, however, is that maximum fluorescence of the reaction product requires an alkaline pH, since 4-MU has a pKa ≈ 8. In an initial screening of five phosphatase substrates based on fluorinated derivatives of 4-MU, all with pKa values lower than that of 4-MU, we found that one substrate, 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP), was much improved for the detection of acid phosphatase activity. When measured at the preferred acid phosphatase reaction pH (5.0), DiFMUP yielded fluorescence signals that were more than 10-fold higher than those of 4-methylumbelliferyl phosphate (MUP). DiFMUP was also superior to MUP for the detection of protein phosphatase 1 activity at pH 7 and was just as sensitive as MUP for the detection of alkaline phosphatase activity at pH 10. A β-galactosidase substrate was also prepared based on 6,8-difluoro-4-methylumbelliferone. This substrate, 6,8-difluoro-4-methylumbelliferyl β-d-galactopyranoside (DiFMUG), was found to be considerably more sensitive than the commonly used substrate 4-methylumbelliferyl β-d-galactopyranoside (MUG), for the detection of β-galactosidase activity at pH 7. DiFMUP and DiFMUG should have great utility for the continuous assay of phosphatase and β-galactosidase activity, respectively, at neutral and acid pH.

Concentration of recombinant baculovirus by cation-exchange chromatography.

BioTechniquesVol. 26, No. 5 BenchmarksOpen AccessConcentration of Recombinant Baculovirus by Cation-Exchange ChromatographyJames BarsoumJames Barsoum*Address correspondence to Dr. James Barsoum, Biogen, Inc., 12 Cambridge Center, Cambridge, MA 02142, USA. Internet: E-mail Address: jim_barsoum@biogen.comBiogen, Cambridge, MA, USASearch for more papers by this authorPublished Online:22 Aug 2018https://doi.org/10.2144/99265bm07AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail FiguresReferencesRelatedDetailsCited ByProduction of Baculovirus and Stem Cells for Baculovirus-Mediated Gene Transfer into Human Mesenchymal Stem Cells22 September 2020Gene Expression in Mammalian Cells Using BacMam, a Modified Baculovirus SystemExpression and purification of recombinant feline interferon in the baculovirus-insect larvae systemProcess Biochemistry, Vol. 49, No. 6Virus Separation Using Membranes20 November 2013Membrane Supported Virus Separation from Biological Solutions14 June 2013 | Chemie Ingenieur Technik, Vol. 85, No. 8Purification of a recombinant baculovirus of Autographa californica M nucleopolyhedrovirus by ion exchange membrane chromatographyJournal of Virological Methods, Vol. 183, No. 2Rational design and optimization of downstream processes of virus particles for biopharmaceutical applications: Current advancesBiotechnology Advances, Vol. 29, No. 6Nonintegrating DNA Virus25 October 2011Baculovirus as vectors for human cells and applications in organ transplantationJournal of Invertebrate Pathology, Vol. 107Quality control and analytical methods for baculovirus-based productsJournal of Invertebrate Pathology, Vol. 107Requirements for baculoviruses for clinical gene therapy applicationsJournal of Invertebrate Pathology, Vol. 107Development and validation of a HPLC method for the quantification of baculovirus particlesJournal of Chromatography B, Vol. 879, No. 1High-titer preparation of Bombyx mori nucleopolyhedrovirus (BmNPV) displaying recombinant protein in silkworm larvae by size exclusion chromatography and its characterization12 June 2009 | BMC Biotechnology, Vol. 9, No. 1Enhancing the multiplication of nucleopolyhedrovirus in vitro by manipulation of the pHJournal of Virological Methods, Vol. 161, No. 2Purification of recombinant baculoviruses for gene therapy using membrane processes2 April 2009 | Gene Therapy, Vol. 16, No. 6Concanavalin a affinity chromatography for efficient baculovirus purificationBiotechnology Progress, Vol. 23Virus Production for Clinical Gene Therapy24 February 2009Targeting and Purification of Metabolically Biotinylated BaculovirusHuman Gene Therapy, Vol. 19, No. 6Baculovirus-Infected Insect Cells Expressing Peptide-MHC Complexes Elicit Protective Antitumor Immunity20 December 2007 | The Journal of Immunology, Vol. 180, No. 1Baculovirus envelope fusion proteins F and GP64 exploit distinct receptors to gain entry into cultured insect cellsJournal of General Virology, Vol. 88, No. 12Ion-Exchange Membrane Chromatography Method for Rapid and Efficient Purification of Recombinant Baculovirus and Baculovirus gp64 ProteinHuman Gene Therapy, Vol. 18, No. 7High yield purification of functional baculovirus vectors by size exclusion chromatographyJournal of Virological Methods, Vol. 142, No. 1-2Sampling Viruses from Soil14 May 2007Gene Expression in Mammalian Cells Using BacMam, a Modified Baculovirus SystemBaculovirus Vectors for Gene TherapyBaculovirus as a highly efficient expression vector in insect and mammalian cellsActa Pharmacologica Sinica, Vol. 26, No. 4Highly efficient baculovirus-mediated gene transfer into rat chondrocytes1 January 2004 | Biotechnology and Bioengineering, Vol. 88, No. 5Generation of chimeric baculovirus with histidine-tags displayed on the envelope and its purification using immobilized metal affinity chromatographyEnzyme and Microbial Technology, Vol. 33, No. 4Baculovirus as Mammalian Cell Expression Vector for Gene Therapy: An Emerging StrategyMolecular Therapy, Vol. 6, No. 1A novel system for the production of fully deleted adenovirus vectors that does not require helper adenovirus22 June 2001 | Gene Therapy, Vol. 8, No. 11Estimation of Baculovirus Titer by β-Galactosidase Activity Assay of Virus PreparationsTetsuro Yahata, Stephanie Andriole, Kurt J. Isselbacher & Toshi Shioda29 August 2018 | BioTechniques, Vol. 29, No. 2Application of Recombinant Baculoviruses in Biopharmaceutical Research Vol. 26, No. 5 Follow us on social media for the latest updates Metrics Downloaded 370 times History Published online 22 August 2018 Published in print May 1999 Information© 2018 Author(s)PDF download

Advanced wastewater disinfection technologies: short and long term efficiency

Advanced disinfection processes (peracetic acid, UV irradiation and ozonation) have been tested and evaluated through bench and pilot scale studies. 3 log removals of total coliforms, faecal coliforms and faecal streptococci were achieved by 10mg/L peracetic acid at a 10min contact time, by UV radiation at 35mW.s/cm2 and by ozone at 5mg/L for 10min contact time. Higher doses are required for virus removal by UV and PAA and especially for highly resistant viruses such as F-specific bacteriophage MS2. Ozonation has the advantage of having a strong effect on all types of bacteriophages and protozoa cysts even when low treatment doses and short contact times are applied. The results of this study demonstrated that evaluation of disinfection efficiency of ozone, UV and PAA depends on the criteria and methods employed. Standard method (plate count) results showed an important disinfection effect on culturability, while results from non-standard methods (respiratory activity and β-galactosidase activity assay) indicated less reduction of viable cells. Moreover, the results confirm that disinfectants act on bacteria in different ways. It has been clearly demonstrated that b-galactosidase activity is affected by PAA while UV treatment has no or very limited effect on the enzyme activity. Even without sunlight reactivation, bacterial regrowth in seawater was observed after disinfection of sewage effluents. This study also shows that the biodegradability of sewage effluent for an E coli strain was affected differently by the oxidative disinfectants ozone and PAA. Biodegradability should therefore be considered when evaluating the total disinfection efficiency.

Cationic polymer and lipids enhance adenovirus-mediated gene transfer to rabbit carotid artery.

Improvement of efficiency of gene transfer to endothelium could be useful for several applications. We tested the hypothesis that cationic nonviral molecules augment adenovirus-mediated gene transfer to blood vessels, perhaps by alteration of the surface charge of adenovirus and facilitation of binding to endothelium. Carotid arteries from rabbits were incubated in vitro for 0.5 to 2 hours with an adenoviral vector alone or noncovalent complexes of adenovirus with poly-L-lysine (a cationic polymer) or lipofectin (a cationic lipid). Binding of adenovirus to the vessels was evaluated immediately after incubation with virus, and assay of transgene (ss-galactosidase) activity and histochemistry were performed 24 hours after gene transfer. To determine whether cationic molecules can be used to augment alteration of vascular function by adenovirus-mediated gene transfer, we also examined effects on gene transfer of endothelial nitric oxide synthase. Assay of ss-galactosidase activity indicated that both cationic molecules increased transgene expression in vessels by approximately 5- to 6-fold. In contrast, when endothelium was removed from the vessels after gene transfer, poly-L-lysine and lipofectin did not significantly increase transgene activity. Histochemistry for ss-galactosidase also suggested that the adenovirus-cationic molecule complexes augmented transgene expression mainly in the endothelium. In addition, we found that complexing adenovirus with cationic molecules increased binding of adenovirus to the vessels. After gene transfer with recombinant adenovirus containing endothelial nitric oxide synthase, calcium ionophore (A23187) produced greater relaxation of vessels treated with adenovirus complexed with poly-L-lysine or lipofectin than those treated with adenovirus alone. Cationic molecules improve the efficiency of adenovirus-mediated gene transfer to blood vessels.

Advanced wastewater disinfection technologies: Short and long term efficiency

Advanced disinfection processes (peracetic acid, UV irradiation and ozonation) have been tested and evaluated through bench and pilot scale studies. 3 log removals of total coliforms, faecal coliforms and faecal streptococci were achieved by 10mg/L peracetic acid at a 10min contact time, by UV radiation at 35mW.s/cm2 and by ozone at 5mg/L for 10min contact time. Higher doses are required for virus removal by UV and PAA and especially for highly resistant viruses such as F-specific bacteriophage MS2. Ozonation has the advantage of having a strong effect on all types of bacteriophages and protozoa cysts even when low treatment doses and short contact times are applied. The results of this study demonstrated that evaluation of disinfection efficiency of ozone, UV and PAA depends on the criteria and methods employed. Standard method (plate count) results showed an important disinfection effect on culturability, while results from nonstandard methods (respiratory activity and β-galactosidase activity assay) indicated less reduction of viable cells. Moreover, the results confirm that disinfectants act on bacteria in different ways. It has been clearly demonstrated that b-galactosidase activity is affected by PAA while UV treatment has no or very limited effect on the enzyme activity. Even without sunlight reactivation, bacterial regrowth in seawater was observed after disinfection of sewage effluents. This study also shows that the biodegradability of sewage effluent for an E coli strain was affected differently by the oxidative disinfectants ozone and PAA. Biodegradability should therefore be considered when evaluating the total disinfection efficiency.

Efficiency of a high-titer retroviral vector for gene transfer into skeletal myoblasts

Background: Genetic transformation of skeletal myoblasts for myocardial repair is dependent on an efficient gene transfer system that integrates the genes of interest into the genome of the target cell and its progeny. The aim of this investigation was to evaluate the use of a new retrovirally based gene transfer system for this purpose. Methods: MFG nlslacZ retroviral vector, packaged in high-titer, split-genome packaging cell line (FLYA4) was used to transduce the skeletal myoblast cell line L6. L6 cells, cultured in 10% fetal calf serum, were transduced with the MFG nlslacZ vector by means of filtered supernatant from FLYA4 cells. Transduced L6 cells were divided into four groups. Group I cells were fixed as myoblasts 3 days after transduction. Group II cells were allowed to differentiate into myotubes. Group III cells were split every 3 days for 4 months. Group IV cells were split as in group III but then allowed to differentiate into myotubes. All samples were fixed and stained for β-galactosidase activity. The effects on gene transfer of transforming growth factor–β, insulin-like growth factor–I, and platelet-derived growth factor were determined by spectrophotometric assay of β-galactosidase activity in cells transduced in the presence or absence of serum with 0 to 200 ng/ml of each growth factor. Results: Morphometric analysis showed that 66.3% ± 3% to 69.6% ± 6% of cells in groups I to IV expressed the lacZ reporter gene. In the presence of serum, transforming growth factor–β significantly inhibited gene transfer, whereas insulin-like growth factor–I and platelet-derived growth factor significantly enhanced gene transfer. In absence of serum, however, only platelet-derived growth factor enhanced retrovirally mediated gene transfer into skeletal myoblasts. Conclusion: MFG retroviral vectors packaged in FLYA4 cells are efficient in gene transfer into skeletal myoblasts and result in transgenic expression that is maintained after repeated cell division, differentiation, or both. Platelet-derived growth factor enhances retrovirally mediated gene transfer into skeletal myoblasts. (J Thorac Cardiovasc Surg 1998;115:1-8)

Early infantile Krabbe disease: deceptively normal magnetic resonance imaging and serial neurophysiological studies

Early infantile Krabbe disease is a progressive neurodegenerative disease caused by deficiency of lysosomal enzyme galactocerebroside β-galactosidase, with onset before the age of 6 months. We present serial clinical, radiological and neurophysiological findings of a patient with early infantile Krabbe disease, presenting at the third day of life with hypotonia, macrocephaly and neonatal seizures. The patient had a deceptively normal initial magnetic resonance imaging examination at the age of 3 months, with progression of the white matter disease over the following 9 months, showing a clinical picture of profound hypotonia with pyramidal and pseudobulbar signs, as well as mild optic atrophy. Assay of galactocerebroside β-galactosidase activity in leukocyte culture disclosed a marked deficiency of the enzyme (0.00 nmol/mg protein per h with normal values >0.7 nmol/mg protein per h), thus confirming the diagnosis of Krabbe disease. Nerve conduction velocity and evoked potential studies, as well as the electroencephalogram, were abnormal at the age of 6 months, while serial neurophysiological studies at the age of 12 and 18 months demonstrated the progressive nature of the disease.

Twist-mediated activation of the NK-4 homeobox gene in the visceral mesoderm of Drosophila requires two distinct clusters of E-box regulatory elements.

NK-4, also called msh2 and tinman, encodes a homeodomain transcription factor that is required for the development of the dorsal mesoderm and its derivatives in the Drosophila embryo. Genetic analyses indicate that NK-4 resides downstream of the mesodermal determinant twist, which encodes a basic helix-loop-helix-type transcription factor. However, the regulation of NK-4 by twist remains poorly understood. Using expression assays in cultured cells and transgenic flies, we show that two distinct clusters of E-box regulatory sequences, present upstream of the NK-4 gene, mediate NK-4 expression in the visceral mesoderm. These elements are conserved between the Drosophila melanogaster and Drosophila virilis NK-4 genes and serve as binding sites for Twist (E1 cluster) and NK-4 (E2 cluster) proteins. In cultured cells, Twist and NK-4 binding results in activation of NK-4 gene expression. In transgenic animals, the E1 and E2 clusters are functionally connected, and both elements are required for NK-4 activation in cells of the visceral mesoderm and also for NK-4 repression in cells of the somatic musculature. These results demonstrate that NK-4 is a direct transcriptional target for Twist and its own gene product in visceral mesodermal cells, supporting the idea that twist and NK-4 function in the subdivision of the mesoderm during Drosophila embryogenesis.

Serial MRI and neurophysiological studies in late-infantile Krabbe disease

We report serial clinical, radiological, and neurophysiological findings of a patient with late-infantile Krabbe disease. At age 13 months, the patient was hospitalized for sudden stiffness and irritability and a diagnosis of spastic diplegia was made. At age 24 months, he was readmitted because of further psychomotor deterioration; neurologically, he manifested severe spastic tetraplegia with optic atrophy. MRI disclosed diffuse high intensity in the cerebral white matter on T 2-weighted images. Nerve conduction velocity and evoked potential studies were markedly abnormal, as were the EEG and the EMG. Assay of galactocerebroside β-galactosidase activity in leukocyte culture disclosed a marked deficiency of the enzyme, confirmatory of the diagnosis of late-infantile Krabbe disease. Serial MRI and neurophysiological studies performed every 6 months for 18 months demonstrated the progressive nature of the disorder, correlating with the clinical deterioration.

Mutation spectrum of 4-nitroquinoline 1-oxide-damaged single-stranded shuttle vector DNA transfected into monkey cells

4-Nitroquinoline 1-oxide (4NQO) is a potent mutagen and carcinogen which induces two main guanine adducts at positions C8 and N2. We recently determined the mutation spectrum induced by the ultimate metabolite of 4NQO, acetoxy-4-aminoquinoline 1-oxide in the M13 lacZ′/E. coli lacZΔM15 α-complementation assay. Our data suggested that dGuo-C8-AQO induces (per se or via AP sites) G to Pyr transversions. Here we report our study on 4NQO mutagenesis in monkey cells. 4NQO lesions were induced in vitro on a single-stranded (ss) DNA shuttle vector carrying the supF tRNA gene. This vector was able to replicate both in mammalian cells and in bacteria. The mutations induced in monkey cells were screened by the white/blue β-galactosidase activity assay in E. coli. We took advantage of the peculiar feature of ss supF DNA in which the extent of secondary structure may be a function of the temperature, with the dependence of the 4NQO-specific adduct spectrum on DNA secondary structure. We reasoned that mutational spectra derived from damage induced in the presence (20°C) or absence (70°C) of DNA secondary structure should be different. The result of sequencing a total of 89 induced and spontaneous mutants confirmed that the spectra are statistically different. These data suggest that the two 4NQO guanine adducts may induce different mutations.

Molecular characterization of yeast regulatory gene CAT3 necessary for glucose derepression and nuclear localization of its product

The yeast regulatory gene CAT3 has an essential function for the derepression of several glucose-repressible enzymes. Therefore, cat3 mutants are unable to grow on maltose or on non-fermentable carbon sources. Unlike the point mutants isolated previously, cat3 null allele strains also failed to utilize raffinose or galactose as sole carbon sources. Sequencing of an 1.6-kb HindIII- BglII fragment complementing cat3 mutations revealed an open reading frame of 322 codons, size of which is in good agreement with the 1.3-kb size of mRNA. No significant similarities with previously sequenced genes could be detected. CAT3- lacZ fusions confirmed the proposed reading frame. A CAT3- lacZ fusion encoding 307 amino acids of CAT3 was able to complement the growth defects of cat3 point mutants and null allele strains. Assay of β-galactosidase activity under different growth conditions indicated a constitutive expression of the CAT3 gene product. Cellular fractionation studies showed the nuclear localization of the CAT3 protein.

Relationship between cellular RecA protein concentration and untargeted mutagenesis in Escherichia coli

We measured the production of untargeted mutations in the cI and cII genes of untreated λ phage undergoing a lytic cycle in UV-irradiated bacterial hosts. As previously shown, treatment with 4 μg/ml of rifampicin during post-irradiation incubation inhibited amplification of the RecA protein in these cells. In addition, we observed a decreased mutation rate compared to the untreated, irradiated bacteria. Treatment with 4 μg/ml or 8 μg/ml rifampicin did not prevent the UV induction of the umuDC operon, as judged by assay of β-galactosidase activity in a umuC-lacZ fusion strain. In contrast, the UV-induction of β-galactosidase in the sulA-lacZ fusion strain was decreased by 4 μg/ml rifampicin. The inhibition of untargeted mutagenesis by this drug treatment was also observed in a strain constitutive for SOS functions ( lexA (Def)) as well as ina RecA-overproducing plasmid strain, that blocks induction of heat-shock proteins, factor(s) in wild-type recA + cells. An htpR165-carrying strain, that blocks induction of heat-shock proteins, exhibited normal UV-promoted mutagenesis. A correlation was observed between the cellular concentration of RecA protein, increased spontaneously by a temperature shift in a lexA(Ts) strain, and the extent of UV-promoted untargeted mutagenesis. These results suggest a mechanistic role of RecA protein in this process.

Time and dose-response study of the effects on rats of the plasticizer di(2-ethylhexyl) phthalate

Groups of male and groups of female Wistar albino rats were administered diets containing sufficient di(2-ethylhexyl) phthalate (DEHP) to ensure intakes of either 1000, 200, or 50 mg/kg/day. Four rats from each experimental group and six control rats of the same sex were killed 3, 7, 14, and 28 days and 9 months after commencement of treatment. At all time points the major abdominal organs were removed and subjected to histological examination. A more extensive necropsy was performed on those rats killed after 9 months of treatment. At all time points the livers of the rats were subjected to extensive histologic, electron microscopic, and biochemical examination. Changes could be grouped according to their time course. Two early and transient alterations were noticed. First, there were morphologic changes in the bile canaliculi of male rats treated with 1000 mg/kg/day of DEHP. Second, there was a burst of mitosis immediately after the start of administration of the compound. The time course of this mitotic burst varied; the increase in mitosis was greatest at 3 days in rats treated with 1000 mg/kg/day of DEHP and was smaller but more prolonged in rats treated with 200 or 50 mg/kg/day. Other changes, namely, a midzonal to periportal accumulation of fat, induction of peroxisomal enzymes, and induction of the P-450 isoenzyme also developed rapidly but were sustained throughout the study. The maximal change was usually attained within 7 days of commencement of treatment. More slowly developing changes were hypertrophy of the hepatocytes, centrilobular loss of glycogen, and a fall in glucose-6-phosphatase activity. Here maximal changes were not attained until 28 days after commencement of treatment. These three effects were clearly observed in rats treated with 200 or 1000 mg/kg/day of DEHP but were only marginally altered in rats treated with 50 mg/kg/day. Finally accumulation of lipid-loaded lysosomes assessed by light and electron microscopy and by assay of β-galactosidase activity was only apparent in rats treated with DEHP for 9 months with 200 or 1000 mg/kg/day of DEHP. Changes in female rats were qualitatively similar to those observed in male rats. The alterations were, however, less pronounced than in male rats treated with an equal dose of DEHP and the degree of liver enlargement was much less because, although the initial hyperplasia was clearly apparent, there was a much smaller degree of hypertrophy.

A microassay for acid β-galactosidase activity toward asialofetuin

To study the enzymatic properties of β-galactosidase from the patients with a β-galactosidase deficiency such as G M1 gangliosidosis, determination of enzymatic activity with naturally occurring substrates, asialofetuin in addition to another natural substrate, G M1 ganglioside, is essentially required. With a previously reported, simple and sensitive fluorometric assay for G M1 ganglioside β-galactosidase using high performance liquid chromatography (HPLC), optimal reaction conditions were determined for the assay of acid β-galactosidase activity toward asialofetuin in skin fibroblast homogenates. Under these conditions, reduced enzymatic activities could be detected in cultured skin fibroblasts from patients with type 1 and 3 G M1 gangliosidoses and mucopolysaccharidosis IV-B (Morquio B syndrome). This method was applicable to study of the enzymatic properties of the mutant β-galactosidase and provided an alternative to assays employing radioactive or artificial substrates.