Mutant Cell Cultures Research Articles

Patient-Derived Cellular Models for Polytarget Precision Medicine in Pantothenate Kinase-Associated Neurodegeneration.

The term neurodegeneration with brain iron accumulation (NBIA) brings together a broad set of progressive and disabling neurological genetic disorders in which iron is deposited preferentially in certain areas of the brain. Among NBIA disorders, the most frequent subtype is pantothenate kinase-associated neurodegeneration (PKAN) caused by pathologic variants in the PANK2 gene codifying the enzyme pantothenate kinase 2 (PANK2). To date, there are no effective treatments to stop the progression of these diseases. This review discusses the utility of patient-derived cell models as a valuable tool for the identification of pharmacological or natural compounds for implementing polytarget precision medicine in PKAN. Recently, several studies have described that PKAN patient-derived fibroblasts present the main pathological features associated with the disease including intracellular iron overload. Interestingly, treatment of mutant cell cultures with various supplements such as pantothenate, pantethine, vitamin E, omega 3, α-lipoic acid L-carnitine or thiamine, improved all pathophysiological alterations in PKAN fibroblasts with residual expression of the PANK2 enzyme. The information provided by pharmacological screenings in patient-derived cellular models can help optimize therapeutic strategies in individual PKAN patients.

Chronic Inflammation Drives p53 Mutant Clonal Hematopoiesis Via Activating the NLRP1 Inflammasome

Individuals with somatic TP53 mutations manifest clonal hematopoiesis and are at high risk of developing hematological malignancies. However, how environmental factors influence the expansion of p53 mutant hematopoietic stem and progenitor cells (HSPCs) is unclear. We found that pI:pC or LPS treatment promotes the clonal expansion of p53 mutant HSPCs in transplantation assays. To determine the impact of LPS on HSPC function in vivo, we treated p53+/+ and p53R248W/+ mice with LPS or PBS and then performed competitive transplantation assays. LPS-treated WT BM cells show decreased engraftment in both primary and secondary transplantation assays, whereas LPS-treated p53 mutant BM cells continued to show increased engraftment. To understand how LPS-induced chronic inflammation promotes p53 mutant HSPC expansion, we examined the impact of mutant p53 on cytokine secretion from BM derived macrophages. We found that p53 mutant macrophages show increased secretion of multiple pro-inflammatory cytokines, including IL-1β, IL-1α, IL-12 (p70), KC, VEGF, IP-10, CXCL-9, LIX, and MCP-1. To further investigate how chronic inflammation promotes p53 mutant HSPC expansion, we performed ATAC-seq assays in LSKs to identify differential regions of chromatin accessibility. Notably, we found that mutant p53 increases the chromatin accessibility to inflammasome genes, including Nlrp1a and Nlrp1b. Inflammasomes are multiprotein complexes that activate Caspase-1 and increase the release of pro-inflammatory cytokines such as IL-1β, leading to caspase-1-dependent death, known as pyroptosis. We found that loss of NLRP1 significantly decreased the secretion of multiple pro-inflammatory cytokines, including IL-1β, IL-1α, IL-6, MCP-1, KC, M-CSF, RANTES, LIX, GM-CSF G-CSF, TNF-α and IL-12 (p40), from macrophages expressing mutant p53. Loss of NLRP1 significantly decreases the repopulating potential of HSPCs expressing mutant p53 in vivo. To further understand how chronic inflammation may confer a competitive advantage to p53 mutant HSPCs, we performed in vitro cell competition assays. We found that WT CD45.1+ cells co-cultured with p53 mutant cells show decreased replating potential compared to WT CD45.1+ cells co-cultured with WT CD45.2+ cells. We measured the levels of cytokines in the condition media utilizing cytokine arrays and found increased levels of IL-1β, IL-6, CXCL-9, and IL-4 in mutant cell cultures compared to that of the WT cells. We found that WT CD45.1+ cells co-cultured with mutant cells show increased pyroptosis. Importantly, we found that the enhanced pyroptosis of WT CD45.1+ cells seen in the mutant group is IL-1β dependent. We found that IL-1β treatment decreases the replating potential of WT BM cells, but not that of p53 mutant cells. To determine the mechanism by which p53 mutant HSPCs are resistant to IL-1β treatment, we performed RNA sequencing in p53+/+ and p53R248W/+ LSKs treated with IL-1β (10 ng/ml) or PBS in vitro for 90 minutes. GSEA analysis revealed that hematopoietic stem and progenitor cell differentiation genes are significantly upregulated in p53 mutant LSKs compared to WT LSKs following IL-1β stimulation. Notably, we found that p53 mutant LSKs treated with IL-1β are enriched with MYC target genes. Furthermore, blocking of IL-1β using IL-1β neutralizing antibody decreases the fitness of p53 mutant HSPCs. Our studies will likely establish both the NLRP1 inflammasome and the pro-inflammatory cytokine IL-1β as novel potential therapeutic targets for preventing clonal hematopoiesis development and treating hematological malignancies with TP53 mutations.

Phenotypic Changes Produced by Endogenous DNA Damage in Yeast Mutants Deficient in Recombination and Base Excision Repair

DNA is under constant attack by exogenous and endogenous factors. Endogenous sources of DNA lesions include free radicals such as reactive oxygen and nitrogen species (RONS), as well as many naturally occurring enzymatic and chemical processes. To prevent mutations and possibly cell death these lesions must be repaired prior to DNA replication. There are five major pathways that are utilized for repair depending on the type of DNA damage. Eukaryotes utilize two pathways, homologous recombination (HR) and nonhomologous end‐joining (NHEJ), for repair of DNA double‐strand breaks (DSBs). In addition, the cells utilize base excision repair (BER) for repair of damaged or missing bases. Previous work in the lab has shown that HR‐deficient yeast mutant cell cultures exhibit high levels of G2 phase cells during normal growth, without exposure to exogenous DNA damaging agents. Inactivation of DNA damage checkpoint genes, but not spindle checkpoint genes, abolished the high G2 cell phenotype, indicating that the damage checkpoint is persistently activated in the cells. In a screen of yeast mutant library strains deficient in each of the major repair pathways, the high G2 phenotype was observed in HR‐ and BER‐deficient cells, but not in mutants specifically defective in nucleotide excision repair (NER), mismatch repair (MMR) or NHEJ.Experiments performed for the current project have assessed the consequences of inactivation of the HR and BER pathways on yeast cells in more detail. The high G2 cell phenotype was heightened in both diploid HR (rad52) and BER (apn1, ogg1) mutants relative to their haploid counterparts. Expression of DIN1 (RNR3), a DNA damage‐inducible gene, was found to be constitutively induced in the HR mutant cells. Preliminary tests of the role of free radicals in generation of endogenous signaling were performed by overexpression of Yap1p, a transcriptional activator of antioxidant enzyme genes. The high G2 cell phenotype was abolished in ogg1 mutants and strongly reduced in rad52 cells when Yap1p was overexpressed. Current work is focused on (a) analysis of DIN1 promoter activation in BER mutants, (b) assessment of the impact of neutralization of free radicals using chemical antioxidants, and (c) measurement of phosphorylated Rad53 checkpoint protein levels using Western blots to characterize the DNA damage checkpoint signaling response.

WHIRLY2 plays a key role in mitochondria morphology, dynamics, and functionality in Arabidopsis thaliana.

WHIRLY2 is a single‐stranded DNA binding protein associated with mitochondrial nucleoids. In the why 2‐1 mutant of Arabidopsis thaliana, a major proportion of leaf mitochondria has an aberrant structure characterized by disorganized nucleoids, reduced abundance of cristae, and a low matrix density despite the fact that the macroscopic phenotype during vegetative growth is not different from wild type. These features coincide with an impairment of the functionality and dynamics of mitochondria that have been characterized in detail in wild‐type and why 2‐1 mutant cell cultures. In contrast to the development of the vegetative parts, seed germination is compromised in the why 2‐1 mutant. In line with that, the expression level of why 2 in seeds of wild‐type plants is higher than that of why 3, whereas in adult plant no difference is found. Intriguingly, in early stages of shoots development of the why 2‐1 mutant, although not in seeds, the expression level of why 3 is enhanced. These results suggest that WHIRLY3 is a potential candidate to compensate for the lack of WHIRLY2 in the why 2‐1 mutant. Such compensation is possible only if the two proteins are localized in the same organelle. Indeed, in organello protein transport experiments using intact mitochondria and chloroplasts revealed that WHIRLY3 can be dually targeted into both, chloroplasts and mitochondria. Together, these data indicate that the alterations of mitochondria nucleoids are tightly linked to alterations of mitochondria morphology and functionality. This is even more evident in those phases of plant life when mitochondrial activity is particularly high, such as seed germination. Moreover, our results indicate that the differential expression of why 2 and why 3 predetermines the functional replacement of WHIRLY2 by WHIRLY3, which is restricted though to the vegetative parts of the plant.

Defects in base excision repair and homologous recombination, but not nucleotide excision repair, lead to altered cell cycle phase distributions in Saccharomyces cerevisiae

The DNA within cells is constantly subjected to chemical modifications resulting from exposure to endogenous and exogenous DNA damaging agents. Damaged or oxidized bases are primarily repaired by the base excision repair (BER) pathway, crosslinks induced by ultraviolet (UV) light and many bulky lesions are repaired by nucleotide excision repair (NER), mismatches are resolved by the mismatch repair system (MMR), and double‐strand breaks in DNA are repaired by either the homologous recombination (HR) pathway or the nonhomologous end‐joining (NHEJ) pathway. Inactivation of NER genes such as RAD2, RAD7 or RAD14 leads to extreme UV sensitivity in yeast cells. Mutants with defects in BER genes OGG1 or UNG1 are defective in repair of oxidized guanines and the removal of uracil bases from DNA, respectively. Yeast rad52 mutants are deficient in HR and are hypersensitive to ionizing radiation and chemicals that induce double‐strand breaks.Our laboratory has previously observed that rad52 haploid and diploid mutant cell cultures have high levels of distended large‐budded G2 phase cells and long cell cycle transit times (see poster by C. England et al.). This work also demonstrated that the high G2 phase cell phenotype of rad52 cells was abolished when any of seven known DNA damage checkpoint genes were co‐inactivated. The current project has extended these studies and asked whether defects in other DNA repair pathways also lead to accumulation of G2 phase cells and increases in doubling times. Initial studies revealed that log phase cultures of mutants deficient in NER (rad2, rad7 and rad14) or MMR (msh2, msh3 and msh6) do not have high levels of large‐budded cells. By contrast, such cells were strongly elevated in mutant cultures defective in the BER pathway (ogg1 and ung1 cells). Recent experiments are attempting to determine the fraction of these large‐budded cells that are G2 vs. M phase cells via DAPI staining and whether they are specifically associated with activation of the DNA damage checkpoint system. Cell cultures containing mutants defective in other DNA repair processes, such as Rad6‐mediated postreplication repair, translesion synthesis and nuclease processing of DNA ends, are also being analyzed using phase contrast and fluorescence microscopy to identify unusual cell morphology and cell cycle phase characteristics.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Clozapine metabolites protect dopaminergic neurons through inhibition of microglial NADPH oxidase.

BackgroundClozapine, an atypical antipsychotic medication, has been effectively used to treat refractory schizophrenia. However, the clinical usage of clozapine is limited due to a high incidence of neutropenia or agranulocytosis. We previously reported that clozapine protected dopaminergic neurons through inhibition of microglial activation. The purpose of this study was to explore the neuroprotective effects of clozapine metabolites clozapine N-oxide (CNO) and N-desmethylclozapine (NDC), as well as their propensity to cause neutropenia.MethodsThe primary midbrain neuron-glia culture was applied to detect the neuroprotective and anti-inflammatory effect of clozapine and its metabolites in lipopolysaccharide (LPS) and MPP+-induced toxicity. And the subsequent mechanism was demonstrated by gp91phox mutant cell cultures as well as microgliosis cell lines. In vivo, to confirm the neuroprotective effect of clozapine and CNO, we measured the dopaminergic neuronal loss and rotarod motor deficits in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-generated mouse Parkinson’s disease (PD) model. The neutropenia or agranulocytosis of clozapine and its metabolites was illustrated by white blood cell count of the treated mice.ResultsWe found that, in midbrain neuron-glia cultures, CNO and NDC were more potent than clozapine in protecting dopaminergic neurons against LPS and MPP+-induced toxicity. CNO and NDC-afforded neuroprotection was linked to inhibition of microglia-mediated neuroinflammation, as demonstrated by abolished neuroprotection in microglia-depleted cultures and their capacity of inhibiting LPS-induced release of proinflammatory factors from activated microglia. NADPH oxidase (NOX2) was subsequently recognized as the main target of CNO and NDC since genetic ablation of gp91phox, the catalytic subunit of NOX2, abolished their neuroprotective effects. CNO and NDC inhibited NOX2 activation through interfering with the membrane translocation of the NOX2 cytosolic subunit, p47phox. The neuroprotective effects of CNO were further verified in vivo as shown by attenuation of dopaminergic neurodegeneration, motor deficits, and reactive microgliosis in MPTP-generated mouse PD model. More importantly, unlike clozapine, CNO did not lower the white blood cell count.ConclusionsAltogether, our results show that clozapine metabolites elicited neuroprotection through inactivation of microglia by inhibiting NOX2. The robust neuroprotective effects and lack of neutropenia suggest that clozapine metabolites may be promising candidates for potential therapy for neurodegenerative diseases.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-016-0573-z) contains supplementary material, which is available to authorized users.

Protection by α-tocopherol of the repair of photosystem II during photoinhibition in Synechocystis sp. PCC 6803

α-Tocopherol is a lipophilic antioxidant that is an efficient scavenger of singlet oxygen. We investigated the role of α-tocopherol in the protection of photosystem II (PSII) from photoinhibition using a mutant of the cyanobacterium Synechocystis sp. PCC 6803 that is deficient in the biosynthesis of α-tocopherol. The activity of PSII in mutant cells was more sensitive to inactivation by strong light than that in wild-type cells, indicating that lack of α-tocopherol enhances the extent of photoinhibition. However, the rate of photodamage to PSII, as measured in the presence of chloramphenicol, which blocks the repair of PSII, did not differ between the two lines of cells. By contrast, the repair of PSII from photodamage was suppressed in mutant cells. Addition of α-tocopherol to cultures of mutant cells returned the extent of photoinhibition to that in wild-type cells, without any effect on photodamage. The synthesis de novo of various proteins, including the D1 protein that plays a central role in the repair of PSII, was suppressed in mutant cells under strong light. These observations suggest that α-tocopherol promotes the repair of photodamaged PSII by protecting the synthesis de novo of the proteins that are required for recovery from inhibition by singlet oxygen.

Proteolytic Processing of the Laminin α3 G Domain Mediates Assembly of Hemidesmosomes but Has No Role on Keratinocyte Migration

Laminin-5 (Lm5), the major adhesion ligand of basal epithelial cells, undergoes complex extracellular proteolytic processing that influences cell adhesion and migration. In tumor cell lines, the proteolytic truncation of the C-terminal G domain of the Lm alpha3 chain induces assembly of hemidesmosomes and downregulates cell migration. To define the biological functions of the alpha3 G domain processing in physiological conditions, we have expressed a series of mutant alpha3 complementary DNA in human primary alpha3-null keratinocytes immortalized by human papillomavirus E6E7 (HKalpha3 cells). Using monolayer and organotypic cell cultures we show that: (1) the hinge region between subdomains G3 and G4 carries the proteolytic cleavage sites; (2) nucleation of the hemidesmosomal proteins is independent of the proteolytic maturation of the alpha3 G domain, whereas formation of mature hemidesmosomes relies on proteolytic cleavage of alpha3; and (3) the proteolytic processing plays no role in cell migration, which suggests that nucleation of hemidesmosomal structures in culture does not reflect the migratory potential of the epithelial cells. Our results also demonstrate that HKalpha3 cells are a unique model system, which will be useful to dissect the functions and molecular interactions of Lm5.

Identification of Cell Cycle-regulated Genes in Fission Yeast

Cell cycle progression is both regulated and accompanied by periodic changes in the expression levels of a large number of genes. To investigate cell cycle-regulated transcriptional programs in the fission yeast Schizosaccharomyces pombe, we developed a whole-genome oligonucleotide-based DNA microarray. Microarray analysis of both wild-type and cdc25 mutant cell cultures was performed to identify transcripts whose levels oscillated during the cell cycle. Using an unsupervised algorithm, we identified 747 genes that met the criteria for cell cycle-regulated expression. Peaks of gene expression were found to be distributed throughout the entire cell cycle. Furthermore, we found that four promoter motifs exhibited strong association with cell cycle phase-specific expression. Examination of the regulation of MCB motif-containing genes through the perturbation of DNA synthesis control/MCB-binding factor (DSC/MBF)-mediated transcription in arrested synchronous cdc10 mutant cell cultures revealed a subset of functional targets of the DSC/MBF transcription factor complex, as well as certain gene promoter requirements. Finally, we compared our data with those for the budding yeast Saccharomyces cerevisiae and found approximately 140 genes that are cell cycle regulated in both yeasts, suggesting that these genes may play an evolutionarily conserved role in regulation of cell cycle-specific processes. Our complete data sets are available at http://giscompute.gis.a-star.edu.sg/~gisljh/CDC.

Antitumour and anticarcinogenic activity of Phyllanthusamarus extract

Aqueous extract of Phyllanthus amarus ( P. amarus) treatment exhibited potent anticarcinogenic activity against 20-methylcholanthrene (20-MC) induced sarcoma development and increased the survival of tumour harboring mice. The extract administration (p.o) was also found to prolong the life span of Dalton's Lymphoma Ascites (DLA) and Ehrlich Ascites Carcinoma (EAC) bearing mice and reduced the volume of transplanted solid tumours. The extract inhibited aniline hydroxylase, a P-450 enzyme. The concentration required for 50% inhibition (IC 50) was found to be 540 μg/ml. The extract was found to inhibit DNA topoisomerase II of Saccharomyces cerevisiae mutant cell cultures and inhibited cell cycle regulatory enzyme cdc25 tyrosine phosphatase (IC 50–25 μg/ml). Antitumour and anticancer activity of P. amarus may be related with the inhibition of metabolic activation of carcinogen as well as the inhibition of cell cycle regulators and DNA repair.

New insights into the assembly of extracellular microfibrils from the analysis of the fibrillin 1 mutation in the tight skin mouse.

The Tight skin (Tsk) mutation is a duplication of the mouse fibrillin 1 (Fbn1) gene that results in a larger (418 kD) than normal (350 kD) protein; Tsk/+ mice display increased connective tissue, bone overgrowth, and lung emphysema. Lung emphysema, bone overgrowth, and vascular complications are the distinctive traits of mice with reduced Fbn1 gene expression and of Marfan syndrome (MFS) patients with heterozygous fibrillin 1 mutations. Although Tsk/+ mice produce equal amounts of the 418- and 350-kD proteins, they exhibit a relatively mild phenotype without the vascular complications that are associated with MFS patients and fibrillin 1-deficient mice. We have used genetic crosses, cell culture assays and Tsk-specific antibodies to reconcile this discrepancy and gain new insights into microfibril assembly. Mice compound heterozygous for the Tsk mutation and hypomorphic Fbn1 alleles displayed both Tsk and MFS traits. Analyses of immunoreactive fibrillin 1 microfibrils using Tsk- and species-specific antibodies revealed that the mutant cell cultures elaborate a less abundant and morphologically different meshwork than control cells. Cocultures of Tsk/Tsk fibroblasts and human WISH cells that do not assemble fibrillin 1 microfibrils, demonstrated that Tsk fibrillin 1 copolymerizes with wild-type fibrillin 1. Additionally, copolymerization of Tsk fibrillin 1 with wild-type fibrillin 1 rescues the abnormal morphology of the Tsk/Tsk aggregates. Therefore, the studies suggest that bone and lung abnormalities of Tsk/+ mice are due to copolymerization of mutant and wild-type molecules into functionally deficient microfibrils. However, vascular complications are not present in these animals because the level of functional microfibrils does not drop below the critical threshold. Indirect in vitro evidence suggests that a potential mechanism for the dominant negative effects of incorporating Tsk fibrillin 1 into microfibrils is increased proteolytic susceptibility conferred by the duplicated Tsk region.

The internal phosphodiesterase RegA is essential for the suppression of lateral pseudopods during Dictyostelium chemotaxis.

Dictyostelium strains in which the gene encoding the cytoplasmic cAMP phosphodiesterase RegA is inactivated form small aggregates. This defect was corrected by introducing copies of the wild-type regA gene, indicating that the defect was solely the consequence of the loss of the phosphodiesterase. Using a computer-assisted motion analysis system, regA(-) mutant cells were found to show little sense of direction during aggregation. When labeled wild-type cells were followed in a field of aggregating regA(-) cells, they also failed to move in an orderly direction, indicating that signaling was impaired in mutant cell cultures. However, when labeled regA(-) cells were followed in a field of aggregating wild-type cells, they again failed to move in an orderly manner, primarily in the deduced fronts of waves, indicating that the chemotactic response was also impaired. Since wild-type cells must assess both the increasing spatial gradient and the increasing temporal gradient of cAMP in the front of a natural wave, the behavior of regA(-) cells was motion analyzed first in simulated temporal waves in the absence of spatial gradients and then was analyzed in spatial gradients in the absence of temporal waves. Our results demonstrate that RegA is involved neither in assessing the direction of a spatial gradient of cAMP nor in distinguishing between increasing and decreasing temporal gradients of cAMP. However, RegA is essential for specifically suppressing lateral pseudopod formation during the response to an increasing temporal gradient of cAMP, a necessary component of natural chemotaxis. We discuss the possibility that RegA functions in a network that regulates myosin phosphorylation by controlling internal cAMP levels, and, in support of that hypothesis, we demonstrate that myosin II does not localize in a normal manner to the cortex of regA(-) cells in an increasing temporal gradient of cAMP.

Effect of Picrorrhiza kurroa extract on transplanted tumours and chemical carcinogenesis in mice

Anti-tumour and anti-carcinogenic activity of Picrorrhiza kurroa extract were studied in mice. Administration of 20-methylcholanthrene (20 MC) produced 100% induction of sarcoma in control mice, whereas the tumour incidence and tumour related deaths were significantly inhibited by the oral administration of P. kurroa extract 150 and 750 mg/kg body weight, respectively. The extract was also found to reduce the volume of transplanted solid tumours induced by Dalton’s lymphoma ascites (DLA) tumour cell lines and increased the life span of ascites tumour bearing mice. P. kurroa extract inhibited yeast topoisomerase I and II enzyme activity when tested on Saccharomyces cerevisiae mutant cell cultures. The extract did not inhibit the enzyme involved in the activation of carcinogen and the cell cycle regulatory enzyme cdc2 kinase.

Participation of mitochondrial proliferation in morphological differentiation of murine mastocytoma cells

Proliferation of a cold-sensitive cell-cycle mutant isolated from an undifferentiated murine mastocytoma line is reversibly arrested at the nonpermissive temperature of 33 °C, and the arrested cells undergo morphological differentiation as expressed by the formation of metachromatic granules. Following transfer of these mutant cells from the permissive temperature of 39.5 to 33 °C, a transient increase in both cytochrome c oxidase and DNA polymerase γ was observed, the ratio of total mitochondrial volume to cell volume nearly doubled within 6 days, and numbers of mitochondrial cross-sections per cellular crosssection as determined in electron micrographs underwent a threefold increase. Addition of chloramphenicol (100 μg/ml) to the mutant cell cultures 6 days prior to transfer from 39.5 to 33 °C prevented the increase in the ratio of total mitochondrial to cell volume. Furthermore, chloramphenicol markedly inhibited the increase in granule number per cell that normally is observed after transfer of cultures to 33 °C or during treatment with 1 m M butyrate. suggesting that mitochondrial proliferation may be an obligatory step in the process of morphological differentiation of these mastocytoma cells.

Type C Niemann-Pick disease. A parallel loss of regulatory responses in both the uptake and esterification of low density lipoprotein-derived cholesterol in cultured fibroblasts.

Low density lipoprotein (LDL) internalization by mutant type C Niemann-Pick (NPC) fibroblasts results in uptake of excess total cholesterol. Uptake of excess lipoprotein cholesterol appears to be mediated by the specific LDL receptor pathway. Associated with excessive LDL-cholesterol uptake is a lesion in early intracellular cholesteryl ester synthesis. In vitro acylCoA:cholesterol acyltransferase activity is normal in cell-free extracts of mutant cells. The ability of exogenous sterols to enhance intracellular esterification of [3H]mevalonate-derived [3H]cholesterol was severely limited in mutant cell cultures suggesting that in vivo activation and/or expression of activated acylCoA:cholesterol acyltransferase may be compromised by the primary mutation of type C Niemann-Pick disease. After 2 days of LDL uptake, rates of intracellular cholesteryl ester synthesis in mutant cells paralleled the rates of esterification in normal cells suggesting that specific early in vivo expression of the acyltransferase may be affected in this disorder.

Presence of the molybdenum-cofactor in nitrate reductase-deficient mutant cell lines of Nicotiana tabacum

Nicotiana tabacum mutant cell cultures lacking nitrate reductase activity were assayed for the presence of the molybdenum-cofactor using its ability to restore NADPH-nitrate reductase activity in extracts of Neurospora crassa nit-1 mycelia. The molybdenum-cofactor of the tobacco wild-type line was shown to complement efficiently the N. crassa nit-1 mutant in vitro. The molybdenum-cofactor seems to exist in a bound form, as acid-treatment was required for release of cofactor activity. Molybdate (5–10 mM), ascorbic acid, and anaerobic conditions greatly increased the activity of the cofactor, demonstrating its high lability and sensitivity to oxygen. Similar results were obtained with two tobacco nia mutants, which are defective in the apoprotein of nitrate reductase. The four cnx mutants studied were shown to contain exclusively an inactive form of the molybdenum-cofactor. This inactive cofactor could be reactivated in vitro and in vivo by unphysiologically high concentrations of molybdate (1–10 mM), thereby converting the cnx cells into highly active cofactor sources in vitro, and restoring nitrate reductase and xanthine dehydrogenase in vivo to partial acitivity. Thus the defect of the cnx mutants resides in a lack of molybdenum as a catalytically active ligand metal for the cofactor, while the structural moiety of the cofactor seems not to be impaired by the mutation. The subunit assembly of the nitrate reductase was found to be independent of the molybdenum content of the cofactor.

Selection and characterization of virus-resistant L cell mutants with changed interferon gene expression.

A study was carried out on the relationship between the antiviral behaviour and changes in interferon (IFN) gene expression of virus-resistant cell mutants. Three virus-resistant L cell mutants were selected by mutagen-selection method. These mutant cell lines were found to show marked changes in their behaviour in both IFN induction and IFN action systems. An accelerated IFN induction response was observed in mutant cell cultures when they were exposed to NDV. Induced mutant cells synthesized IFN mRNA more quickly, and the IFN titers after induction with poly I:C were higher in mutant cell lines than those in the parent cell line. The virus-resistant L cell mutants appeared to be more sensitive to the anticell-division activity caused by ppp A2'p5'A2'p5'A. The results obtained suggest that changes in IFN gene expression may account for the antiviral behaviour of these cell mutants.

Gene Expression in Cultured Mammalian Cells

Publisher Summary This chapter discusses the gene expression in cultured mammalian cells. Cell and tissue culture provides a simple system for investigating a variety of fundamental problems involved in gene expression. Cells can be grown under relatively controlled environmental conditions that permit analysis of the physiology, biochemistry, and genetics of cells in culture. New methods of cell fusion and hybridization allow some aspects of recombination analysis in mammalian cell culture, and the recent development of methods for enucleating mammalian cells with the recovery of cytoplasm and nuclei separately permits reconstruction of cells by fusing nuclear and cytoplasmic components derived from different cell types. The chapter emphasizes on mutant cell cultures, as well as examples of enzyme regulation and metabolic control that reflect gene expression in cultured mammalian cells. Studies on mutant mammalian cells, particularly those that show temperature sensitivity toward expressing differentiated activities, may be useful in separating and analyzing the required biochemical events. Difficulty in selecting for such mutant cells is a serious limitation, but recent studies on cell lines selected for temperature-sensitive differences in contact inhibition of cell growth confirm the resolving power of such methods. Cell lines derived from mutant subjects and from multipotential tumors are particularly informative for investigating basic mechanisms of gene expression and determination. Cell fusion and the subsequent analysis of complementation in heterokaryons and synkaryons may provide insight into the nature of mutations at the levels of the defective protein and interactions of the regulatory process.

Purine mutants of mammalian cell lines. I. Accumulation of formylglycinamide ribotide by purine mutants of Chinese hamster ovary cells.

Mutants unable to perform de novo biosynthesis of purines have been isolated from cultures of mutagen-treated Chinese hamster ovary cells using bromodeoxyuridine selection techniques. Accumulation of C14-labeled formylglycinamide ribotide by suspension cultures of mutant cells incubated with glycine-C14 suggested that the defect leading to auxotrophy most probably involves the gene coding for formylglycinamide amidotransferase, (E.C. 6.3, 5.3), the fourth enzyme in the de novo purine biosynthetic pathway. Direct assay of formylglycinamide amidotransferase activity in cell-free extracts prepared from mutant and parental cells has demonstrated the absence of amidotransferase activity in mutant derived extracts.

Antibiotic resistant group A streptococci. 3. Penicillin resistance and mitomycin C inactivation of mutants which lose soluble hemolysin production.

ABSTRACTGroup A streptococcus mutants, which lost the ability to produce soluble hemolysins, either β or α hemolytic on the horse blood agar, showed penicillin‐mitomycin C double resistance. Penicillin resistance was high and stable, but mitomycin C resistance was not remarkably high. Mitomycin C resistance was always accompanied with inactivation of the antibacterial activity of the drug, while mitomycin C and penicillin sensitive group A streptococci never showed mitomycin inactivation. The component affecting the mitomycin C was not found in the sterile filtrate of mutant cell cultures, nor could it be extracted by sonic oscillation. The activity of the factor diminished, when living mutant cells were heated at 60 C for 30 min, but activity was not diminished at 56 C. The intensity of activity was a function of the bacterial quantities used. Mutant cells suspended in heart infusion broth, proteose peptone, casamino acid and horse serum were able to render the factor which inactivated mitomycin C, while mutant cells suspended in bovine serum albumin, RNA, or saline did not show the phenomenon.