Β-galactosidase Activity In Cells Research Articles

Abstract 136: Phospho-S6 levels correlate with response to Copanlisib (BAY 80-6946) in multiple myeloma

Abstract Background: Copanlisib (BAY 80-6946) is a reversible, pan-class I PI3K inhibitor with preferential activity for the alpha isoform, which may be of particular importance in multiple myeloma. Here we demonstrated the in vitro efficacy of copanlisib in a panel of 20 multiple myeloma cell lines. Baseline levels of phospho-S6 (P-S6) correlated with sensitivity to copanlisib, resulting in a potential biomarker of response. In addition, the change of P-S6 post-treatment could be used as a pharmacodynamic biomarker for copanlisib treatment. Methods: We screened a panel of 20 multiple myeloma cell lines and selected 3 sensitive: NCI-H929, MM.1S, L-363, and 3 resistant: AMO-1, JJN3, COLO-677 for further analysis. We performed apoptosis and cell senescence assays following 72 hours of 50nM and 100nM copanlisib exposure. Cell cycle analysis and induction of apoptosis were performed by FACS after propidium iodide or PI/ANX-V FITC staining, respectively. Cellular senescence was determined by measuring β-galactosidase activity in cells treated for 96 hours. Reverse phase protein array (RPPA) was performed at baseline and post treatment for proteomic analysis with confirmatory western blots. Flow cytometry was also performed to monitor the post-treatment P-S6 level changes. Results: Copanlisib treatment induced apoptosis in the sensitive cell lines (50-80% AN-V+ cells) but not in resistant cell lines (1-5% AN-V+ cells). An increased cell cycle arrest in G1 was also observed in the sensitive cell lines but not in the resistant lines. The cell senescence assays confirmed apoptosis rather than cell senescence as the mechanism of inhibition of proliferation. RPPA analysis demonstrated lower baseline p-S6 (S235/236, S240/244) protein levels in sensitive compared to resistant cell lines and this was confirmed with western blot analysis. Treatment with copanlisib resulted in a greater decrease in p-S6 in the sensitive cell lines NCI-H929 and L363 (53-83%, 73-93% respectively) than in the resistant cell lines COLO-677 and JJN3 (5-27%, and 38-67%, respectively), which was validated by western blot and phospho-flow. We also showed by RPPA and WB that copanlisib down-regulates pro-survival and proliferation molecules including p-S6K1, p-S6 and p-4EBP1, and upregulates pro-apoptotic PDCD4 in all cell lines, but to a greater extent in sensitive cell lines. Finally, pharmacodynamic p-S6 response remained at different post-treatment time points. Discussion: A differential response to copanlisib is seen in the myeloma cell line panel. A subgroup of multiple myeloma cell lines demonstrated median IC50 values in the low nanomolar range (5-100nM), and responses correlated with low baseline P-S6. This p-S6 stratified response was only observed with PI3K-alpha inhibitors, but not with inhibitors targeting other PI3K isoforms or pan-PI3K inhibitors. Further studies may allow development of a new patient screening method or companion diagnostic. Citation Format: Sarah Larson, Mao Yu Peng, Monica Mead, Andrae Vandross, Dylan Conklin, Erika Von Euw, Dennis J. Slamon. Phospho-S6 levels correlate with response to Copanlisib (BAY 80-6946) in multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 136. doi:10.1158/1538-7445.AM2017-136

Activity of the Phosphatidylinositol 3-Kinase (PI3K) Inhibitor, Copanlisib, As a Single Agent and in Combination with Carfilzomib in Multiple Myeloma

Background: The PI3K pathway signals for cell proliferation and survival in many malignancies including multiple myeloma. Copanlisib (BAY 80-6946) is a pan-class I PI3K inhibitor with preferential activity of the alpha and delta isoforms, of which the alpha isoform has particular importance in multiple myeloma. Here we demonstrate the pharmacological activity of copanlisib in multiple myeloma as a single agent and in combination with carfilzomib biomarker exploratory evaluation using phosphorylation of the S6 ribosomal protein (p-S6).Methods: 21 multiple myeloma cell lines were initially screened. Using an IC50 cut off of 100nM, 3 sensitive: NCI-H929, MM.1S, L-363 and 3 resistant: AMO-1, JJN3, COLO-677 were selected for further analysis. Apoptosis and cell senescence assays were done with each agent (copanlisib at 50nM and 100nM at 72 hours; carfilzomib at 2 nM and 20nM at 96 hours). Cell cycle analysis and induction of apoptosis were performed by FACS after propidium iodide or Annexin V FITC staining, respectively. Cellular senescencewas determined by measurement of β-galactosidase activity in cells treated for 96 hours. Combination studies utilized excess over highest single agent statistics (EOHSA) to evaluate potentiation. Reverse phase protein array (RPPA) was performed at baseline and post treatment for proteomics analysis with confirmatory western blot at 4 and 24 hours post treatment.Results: Copanlisib induced apoptosis and cell cycle arrest in the sensitive cell lines, but not the resistant cell lines. The cell senescence assays confirmed apoptosis rather than cell senescence as the mechanism of inhibition of proliferation. Pretreatment RPPA analysis demonstrated lower p-S6 levels in the sensitive cells lines compared to the resistant cell lines. Further, treatment with copanlisib resulted in a greater decrease in p-S6 in the sensitive cell lines than in the resistant cell lines, which was validated by western blot. Downstream pathway effects were confirmed by an increase in PDCD4 in the sensitive cell lines. Treatment with copanlisib and carfilzomib showed potentiation by EOHSA statistics and further decrease in p-S6 expression in the sensitive rather than resistant cell lines.Discussion: Copanlisib demonstrated single agent activity in human multiple myeloma cell lines, which is enhanced by the addition of carfilzomib. p-S6 levels may serve to select the most appropriate patient population to study combination of carfilzomib and copanlisib in relapsed/refractory multiple myeloma. With the choices of therapy available to patients with multiple myeloma there is a need for predictive biomarkers in order to better sequence therapies. DisclosuresLarson:BMS: Consultancy. Slamon:Novartis: Consultancy, Honoraria, Research Funding; Biomarin: Consultancy, Honoraria; Pfizer: Honoraria, Research Funding; Eli Lilly: Consultancy; Syndax: Research Funding; Bayer: Consultancy.

Efficacy and Mechanism of Action of the Novel Bromodomain Inhibitor, PLX51107, in B Cell Malignancies

Background: BRD4 functions as an important epigenetic regulator of transcription of pathways commonly involved in oncogenesis, including cell cycle progression and apoptosis. Inhibition of BRD4 with small molecule inhibitors has demonstrated activity in various hematologic and solid malignancies. While downregulation of c-myc following BRD4 inhibition has been described, the exact mechanism of action of BRD4 inhibitors across B cell malignancies remains unclear. Here we demonstrate the efficacy of PLX51107 across a spectrum of B cell malignancies and the signaling pathways involved.Methods: A panel of 22 human multiple myeloma (MM) cell lines and 56 B cell lymphoma (BCL) cell line, were evaluated for the effects of PLX51107 on cell proliferation after a 5 day exposure to the drug. Sensitive NCI-H929, MM.1S, KMS-12BM and resistant RPMI, EJM, L363 - MM cell lines were selected for further mechanism of action (MOA) studies; along with BCL sensitive DoHH2- DLBCL GCB subtype, Ly3- DLBCL ABC subtype, MINO-mantle cell, 2A8-Burkitt. Matched resistant cell lines SUDHL-8, U2932, GRANTA, DB were also selected for the MOA studies. All MOA assays were performed by treating the MM cells at a dose of 150nM PLX51107 except the BCL cells which were exposed to 300nM. Cell cycle analysis and induction of apoptosis was performed by FACS after propidium iodide or Annexin V FITC staining respectively on cells treated for 72 hours. Cellular senescencewas determined by measurement of β-galactosidase activity in cells treated for 7 days (BCL) or 5 days (MM). Western blot analyses for c-myc, p-IRAK, p-Iκβα, p65 and p-MAPK Erk1/2 were performed at 4 and 24 hours post treatment. DMSO treatment was included as a control in the MOA experiments.Results: PLX51107 potently inhibited the in vitro proliferation and survival of a variety of B cell malignancies, including GCB and ABC subtype DLBCL, mantle cell lymphoma, Burkitt lymphoma, and multiple myeloma cell lines.Cell cycle arrest in G1 phasewas observed in 3/4 sensitive BCL, Ly3, 2A8 and MINO and 2/3 sensitive MM, NCI-H2929, MM1.S. Increased apoptosis was demonstrated in the sensitive cell lines 2A8, MINO (BCL) and NCI-H2929, KMS12BM (MM). Three sensitive BCL (DoHH2, Ly3, 2A8) and 2 sensitive MM (KMS12BM, MM1.S) displayed premature senescence. Interestingly, DoHH2 (DLBCL-GCB), Ly3 (DLBCL-ABC) and the MM cell line MM1.S, all of which were growth inhibited by PLX51107 treatment, did not demonstrate an increase in apoptosis and all were found to have the greatest degree of premature senescence. Our data indicate that these differential mechanistic findings do not correspond with a known underlying predictive biomarker. In contrast to the resistant cell lines, all sensitive lines demonstrated marked downregulation of c-myc expression after PLX51107 treatment. Downregulation of p-IRAK, a signaling intermediary of the MyD88-dependent pathway, was also observed in all sensitive BCL cell lines, while no corresponding change of this marker was observed in the sensitive MM cell lines. P-MAPK Erk 1/ 2 was downregulated in all sensitive BCL cell lines, but only 1 of 3 sensitive MM cell lines. Expression of p-Iκβα, a regulator of the NFκʙ pathway, was unchanged in all sensitive BCL cell lines but was downregulated in all 3 sensitive MM cell lines.Discussion: PLX51107 demonstrates in vitro efficacy through differential mechanisms across a maturational spectrum of B-cell malignancies. Treatment of BCL and MM cell cultures results in downregulation of c-myc in sensitive cell lines, consistent with findings from previous BRD4 inhibitors. We demonstrate the involvement of novel signaling pathways with PLX51107 treatment that vary based on the maturational stage of the malignant B cell. These data suggests treatment with PLX51107 affects the MyD88-dependent toll-like receptor pathway in the BCL cell cultures, while the NFκʙ pathway is changed in MM cell cultures. In conclusion, these results demonstrate that PLX51107 has distinct mechanisms of action beyond downregulation of the c-myc transcriptional program and is a promising targeted therapeutic agent across a broad range of clinically and histologically diverse B-cell neoplasms. DisclosuresPowell:Plexxikon: Employment. Slamon:Pfizer: Equity Ownership, Research Funding; BioMarin: Membership on an entity's Board of Directors or advisory committees, Research Funding. Larson:BMS: Consultancy.

Pikfyve Is Essential for Lysosomal Homeostasis in Macrophages

The biogenesis and degradative functions of the lysosome in macrophages and other cells is critically dependent on the membrane trafficking along the endolysosomal system. This trafficking is tightly regulated by several signaling molecules including the endosomal phosphoinositide PtdIns(3,5)P2, which is synthesized from PtdIns(3)P in mammals by the lipid kinase, PIKfyve. Although PtdIns(3,5)P2 deficiency is associated with neurodegeneration in mice and humans, the critical importance of PIKfyve to normal macrophage function is poorly understood. To address this, we genetically engineered mice to lack PIKfyve in their macrophages by pairing PIKfyvefl/fl mice with mice that are transgenic for the myeloid-specific LysM-Cre (PIKfyvefl/fl LysM-Cre). PIKfyvefl/fl LysM-Cre mice develop weight gain and massive hepatosplenomegaly. Immunohistochemical analyses of the liver and spleen from PIKfyvefl/fl LysM-Cre mice demonstrate tissue infiltration of mononuclear cells with numerous cytoplasmic vacuoles that stain with the macrophage-specific marker, anti-F4/80 antibody. To verify that these vacuolated cells result from PIKfyve ablation, we utilized genetic lineage tracing by crossing PIKfyvefl/fl LysM-Cre mice with mice transgenic for a Cre-dependent β-galactosidase reporter. Histochemical analyses using a chromogenic substrate, x-gal, reveal β-galactosidase activity in cells with small cytoplasmic vacuoles, demonstrating Cre expression and therefore PIKfyve ablation in these cells. However, β-galactosidase activity is absent in cells with large vacuoles, indicating that these cells do not express Cre and still contain PIKfyve. These findings suggest that the cytoplasmic vacuolation resulted from both cell-autonomous and non-cell-autonomous effects of PIKfyve deletion. To investigate the macrophage-specific effects of PIKfyve ablation in the lysosomal biogenesis and functions, we isolated macrophages from the bone marrow and spleen of PIKfyvefl/fl LysM-Cre mice using magnetic beads bound to anti-F4/80 antibody. Immunoblot analysis of F4/80+ PIKfyve-null macrophages reveal increased expression of lysosomal proteins including Cathepsin D, Cathepsin S, and LAMP-1, demonstrating that PIKfyve is essential for lysosome homeostasis in macrophages.Recent studies showed that the transcription factor TFEB is a master regulator of lysosomal biogenesis and function. When TFEB is phosphorylated by mTORC1 on the lysosomal membrane, TFEB is retained in the cytosol, and therefore unable to promote gene transcription. However, when TFEB is de-phosphorylated, TFEB translocates to the nucleus where it is transcriptionally active. Since PtdIns(3,5)P2 was previously shown to regulate the recruitment of mTORC1 to the lysosome, we hypothesized that the synthesis of PtdIns(3,5)P2 by PIKfyve regulates TFEB phosphorylation and indirectly controls TFEB transcriptional activity. To test this hypothesis, we studied the expression of TFEB in the F4/80+ macrophages isolated from the control and PIKfyvefl/fl LysM-Cre mice. While wild-type macrophages express TFEB in both phosphorylated and dephosphorylated forms, PIKfyve-null macrophages primarily express the dephosphorylated form of TFEB. This suggests that PIKfyve is required for the phosphorylation of TFEB and consequently PIKfyve prevents TFEB’s ability to transcribe lysosomal genes. Thus, PIKfyve ablation within macrophages upregulates the transcription of lysosomal genes via TFEB activation. Our study suggests that PIKfyve is a critical regulator of lysosomal homeostasis and demonstrates a previously unrecognized link between PIKfyve, PtdIns(3,5)P2 synthesis, and the TFEB pathway in the regulation of lysosomal biogenesis and function within macrophages. DisclosuresNo relevant conflicts of interest to declare.

Protonation-mediated structural flexibility in the F conjugation regulatory protein, TraM

TraM is essential for F plasmid-mediated bacterial conjugation, where it binds to the plasmid DNA near the origin of transfer, and recognizes a component of the transmembrane DNA transfer complex, TraD. Here we report the 1.40 A crystal structure of the TraM core tetramer (TraM58-127). TraM58-127 is a compact eight-helical bundle, in which the N-terminal helices from each protomer interact to form a central, parallel four-stranded coiled-coil, whereas each C-terminal helix packs in an antiparallel arrangement around the outside of the structure. Four protonated glutamic acid residues (Glu88) are packed in a hydrogen-bonded arrangement within the central four-helix bundle. Mutational and biophysical analyses indicate that this protonated state is in equilibrium with a deprotonated tetrameric form characterized by a lower helical content at physiological pH and temperature. Comparison of TraM to its Glu88 mutants predicted to stabilize the helical structure suggests that the protonated state is the active form for binding TraD in conjugation.

Nonsense Suppression in Yeast Cells Overproducing Sup35 (eRF3) Is Caused by Its Non-heritable Amyloids

The [PSI+] prion determinant of Saccharomyces cerevisiae causes nonsense suppressor phenotype due to a reduced function of the translation termination factor Sup35 (eRF3) polymerized into amyloid fibrils. Prion state of the Rnq1 protein, [PIN+], is required for the [PSI+] de novo generation but not propagation. Yeast [psi-] [PIN+] cells overproducing Sup35 can exhibit nonsense suppression without generation of a stable [PSI+]. Here, we show that in such cells, most of Sup35 represents amyloid polymers, although the remaining Sup35 monomer is sufficient for normal translation termination. The presence of these polymers strictly depends on [PIN+], suggesting that their maintenance relies on efficient generation de novo rather than inheritance. Sup35 polymers contain Rnq1, confirming a hypothesis that Rnq1 polymers seed Sup35 polymerization. About 10% of cells overproducing Sup35 form colonies on medium selective for suppression, which suggests that the proportion of Sup35 monomers to polymers varies between cells of transformants, allowing selection of cells deficient for soluble Sup35. A hybrid Sup35 with the N-terminal domain replaced for 66 glutamine residues also polymerizes and can cause nonsense suppression when overproduced. The described polymers of these proteins differ from the [PSI+] polymers by poor heritability and very high frequency of the de novo appearance, thus being more similar to amyloids than to prions.

UGA4 gene encoding the γ-aminobutyric acid permease in Saccharomyces cerevisiae is an acid-expressed gene

Background and aims: biological processes in all organisms are controlled by environmental conditions, however, information concerning the molecular responses to external pH is scarce. In this work we studied the pH response of UGA4 gene encoding δ-aminolevulinic acid and γ-aminobutyric acid permease in Saccharomyces cerevisiae. Methods: we analyzed the effect of pH on the expression of UGA4 gene measuring β-galactosidase activity in cells carrying a UGA4::lacZ fusion gene. Results: results indicate that UGA4 expression is higher at acidic pH. The expression of UGA3 and UGA35 genes, which encode two positive transcription factors, is not regulated by external pH, while the expression of UGA43 gene encoding a repressor of UGA4 transcription is dependent on pH. Using a strain lacking Uga43p we clearly showed that the effect of ambient pH on UGA4 expression is not a secondary effect of the pH regulation on UGA43. We have also demonstrated that the effect of pH can only be detected when UGA4 gene is not subject to a strong repression by Uga43p nor to GABA induction. Conclusion: here, we demonstrate that UGA4 is an acid-expressed gene. This regulation is probably mediated by Rim101p through the consensus site 5′-GCCARG-3′ at 237 bp preceding the UGA4 coding sequence (201).

Medial Edge Epithelial Cell Fate during Palatal Fusion

To explain the disappearance of medial edge epithelial (MEE) cells during palatal fusion, programmed cell death, epithelial–mesenchymal transformation, and migration of these cells to the oral and nasal epithelia have been proposed. However, MEE cell death has not always been accepted as a mechanism involved in midline epithelial seam disappearance. Similarly, labeling of MEE cells with vital lipophilic markers has not led to a clear conclusion as to whether MEE cells migrate, transform into mesenchyme, or both. To clarify these controversies, we first utilized TUNEL techniques to detect apoptosis in mouse palates at the fusion stage and concomitantly analyzed the presence of macrophages by immunochemistry and confocal microscopy. Second, we in vitro infected the MEE with the replication-defective helper-free retroviral vector CXL, which carries the Escherichia coli lacZ gene, and analyzed β-galactosidase activity in cells after fusion to follow their fate. Our results demonstrate that MEE cells die and transform into mesenchyme during palatal fusion and that dead cells are phagocytosed by macrophages. In addition, we have investigated the effects of the absence of transforming growth factor β3 (TGF-β3) during palatal fusion. Using environmental scanning electron microscopy and TUNEL labeling we compared the MEE of the clefted TGF-β3 null and wild-type mice. We show that MEE cell death in TGF-β3 null palates is greatly reduced at the time of fusion, revealing that TGF-β3 has an important role as an inducer of apoptosis during palatal fusion. Likewise, the bulging cells observed on the MEE surface of wild-type mice prior to palatal shelf contact are very rare in the TGF-β3 null mutants. We hypothesize that these protruding cells are critical for palatal adhesion, being morphological evidence of increased cell motility/migration.

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)

Spacing of promoter elements regulates the basal expression of the soxS gene and converts SoxR from a transcriptional activator into a repressor.

SoxR protein of Escherichia coli governs a global response against superoxide-generating agents (such as paraquat) or nitric oxide, and provides broad antibiotic resistance. A redox signal activates SoxR post-translationally to trigger transcription of a second regulatory gene, soxS. Activated and non-activated SoxR bind the soxS promoter with the same high affinity, but only the activated protein stimulates soxS transcription. SoxR acts by an unusual mechanism of positive control: the protein binds the soxS promoter between near-consensus -10 and -35 elements that are separated by an unusually long 19 bp (versus the optimal 17 bp). We have constructed and analyzed site-specific deletions that alter the promoter element spacing. Reducing the spacer length to 16-18 bp dramatically elevated basal expression of soxS in vivo and in vitro, and nearly eliminated additional activation by SoxR in response to paraquat. More strikingly, shortening the spacer converted SoxR from an activator into a repressor regardless of paraquat treatment. Gel mobility-shift assays show that repression by SoxR of the promoters with 17 and 16 bp spacers is due to interference with binding by RNA polymerase. Thus, activated SoxR remodels the unusual configuration of the wild-type soxS promoter into a highly active form, probably by compensating for the suboptimal distance between the -10 and the -35 elements.

Adenovirus-mediated gene transfer to normal and atherosclerotic arteries. A novel approach.

Previous studies of gene transfer to blood vessels in vivo have relied on intraluminal, catheter-based methods for delivery of adenoviral and other vectors. In this study, topical application of a replication-deficient adenoviral vector was used as an alternative method of gene transfer to the vessel wall. We administered recombinant adenovirus (1.0 x 1.5 x 10(10) pfu/mL) containing the nuclear targeted bacterial beta-galactosidase gene topically to arteries in normal and atherosclerotic cynomolgus monkeys. Topical administration was achieved by injection of adenoviral suspension within the periarterial sheath. Segments of femoral and carotid arteries were examined histochemically after staining with 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside 1 day after treatment with the adenovirus. After topical administration of virus, beta-galactosidase activity was observed in approximately 20% of cells in the adventitia in both normal and atherosclerotic arteries. There was no detectable beta-galactosidase activity in cells of the intima or media. Thus, topical application provides an alternative method for gene transfer to blood vessels in vivo. This approach, which does not require interruption of blood flow and does not disrupt the endothelium, may be useful for studies of vascular biology and perhaps gene therapy in both normal and atherosclerotic vessels.

Expression of the recA gene is reduced in Escherichia coli topoisomerase I mutants

We studied the influence of DNA topological changes on Escherichia coli recA gene expression. This was monitored by measuring β-galactosidase activity in cells containing a recA-lacZ fusion. To modulate DNA supercoiling we used mutations in the genes encoding for topoisomerase I and DNA gyrase. After either UV irradiation or treatment with the gyrase inhibitor ciprofloxacin, induction of the recA gene was reduced in topA10 mutants, this reduction being alleviated when gyrA or gyrB mutations causing DNA relaxation were present. A reduced induction of recA was also observed after incubation of cells carrying the recA441 mutation at 42°C in the presence of adenine. Using bacteria deficient in the LexA repressor, we have demonstrated that the topA10 mutation reduces the constitutive expression of the recA gene. We suggest that the increase in negative supercoiling resulting from topoisomerase I deficiency interferes with transcription from the recA promoter. The reduction in the expression of the recA gene in topA10 bacteria could determine their increased UV sensitivity as well as their partial defectiveness in SOS mutability.

Hydrolysis of whey by whole cells of Kluyveromyces bulgaricus immobilized in calcium alginate gels and in hen egg white

Whey hydrolysis was compared in column reactors containing whole yeast cells immobilized in Ca-alginate or in hen egg white in relation to cell β-galactosidase activity, flow rates, temperature and time. With cells of 1.3 U/mg dry weight (ONPG method) immobilized in Ca-alginate, 80% hydrolysis was obtained at 4° and 20° C with, respectively 0.50 and 1.65 bed volume/H; the values were 0.2 and 0.74 with cells entrapped in hen egg white. When the flow rate was expressed as ml/H/g wet yeast, no significant difference was observed between both matrices and 80% hydrolysis was reached with a flow rate 1.7 and 5 according to the temperature. The best performance was achieved by the yeast egg white reactor. At 4°C, hydrolysis decreased by 10% after 13 days; by 20% after 17 days. The presence of lactose transport inhibitors in whey did not significantly influence lactose hydrolysis.

Yeast cells with high β-galactosidase activity and their immobilization

The batch growth conditions of K. lactis were optimized for high β-galactosidase content using a modified inorganic salt medium with 1% lactose at 30 °C and pH as an indicator for the harvest time. Cell β-galactosidase activity of up to 966 LAU (g cell) −1 was achieved when the cell wall was permeabilized by Triton. The cells in batch operation tended to utilize glucose when it accumulated rather than lactose. The immobilized cells should be therefore used in a continuous plug flow reactor. The experiments with a 50 ml column reactor filled by the cells immobilized in polyurethane foam showed a good activity with no leakage of cells and a half-life time of approximately 3 d.

Regulated OmpF porin synthesis in Escherichia coli K-12

The effects of growth conditions on the expression of the Escherichia coli K-12 major outer membrane porin gene ompF were determined by measuring the amount of OmpF protein incorporated into the outer membrane, β-galactosidase activity in cells in which lacZ was regulated by the ompF promoter, and by studying their effects in the presence of the general transcription inhibitor rifampicin. A variety of conditions reduced ompF transcription. Common features among the inhibitors of ompF expression could not be identified; another protease inhibitor and another uncoupler did not specifically affect ompF expression. It is therefore concluded that the inhibitors interact directly or indirectly with a sensory system (possibly EnvZ protein) which affects the porin gene regulon accordingly.

Regulated OmpF porin synthesis inEscherichia coliK-12

The effects of growth conditions on the expression of the Escherichia coli K-12 major outer membrane porin gene ompF were determined by measuring the amount of OmpF protein incorporated into the outer membrane, β-galactosidase activity in cells in which lacZ was regulated by the ompF promoter, and by studying their effects in the presence of the general transcription inhibitor rifampicin. A variety of conditions reduced ompF transcription. Common features among the inhibitors of ompF expression could not be identified; another protease inhibitor and another uncoupler did not specifically affect ompF expression. It is therefore concluded that the inhibitors interact directly or indirectly with a sensory system (possibly EnvZ protein) which affects the porin gene regulon accordingly.

Acid glycosidases in mucopolysaccharidoses fibroblasts

β-Galactosidase, β- N-acetyl glucosaminidase, β-glucuronidase, and glucose 6-phosphate dehydrogenase activities have been determined in extracts of cultured fibroblasts from individuals with the Type I, II, and III mucopolysaccharidoses, their parents, and normal controls. A wide range of specific activity was encountered for each of the enzymes with extensive overlap between all experimental groups. The only significant difference detected was an approximately 50% depression in the mean β-galactosidase activity in cells from mucopolysaccharidoses patients. The electrophoretic pattern of the β-galactosidase activity in fibroblasts from patients showed the presence of all of the forms seen in normal cells. These results along with other lines of reasoning, lead to the conclusion that the β-galactosidase defect seen in tissues from mucopolysaccharidoses patients, Type I, II, and III is probably not the primary genetic lesion.