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PubMed1994

The Yak1 protein kinase of Saccharomyces cerevisiae moderates thermotolerance and inhibits growth by an Sch9 protein kinase-independent mechanism.

The growth defect associated with the loss of yeast A kinase activity can be alleviated by the overexpression or deletion of two other kinases, Sch9 and Yak1, respectively. Using tests of epistasis, we have shown that Sch9 and Yak1 define separate signaling pathways and must, therefore, suppress the A kinase defect by different mechanisms. Nevertheless, the Yak1 kinase appears to regulate cellular processes that are under A kinase control. For example, acquisition of heat resistance is correlated with Yak1 kinase activity, such that YAK1-overexpressing cells are over 200-fold more resistant than isogenic yak1 strains. These results, for the first time, associate a phenotype, other than suppression of the A kinase growth defect, with the loss of Yak1 activity and argue a broader role for the Yak1 kinase in cell growth.

Cell Cycle↗
PubMed2006

Mapping a sex hormone-sensitive gene determining female resistance to liver carcinogenesis in a congenic F344.BN-Hcs4 rat.

Hepatocellular carcinoma (HCC) is prevalent in human and rodent males. Hepatocarcinogenesis is controlled by various genes in susceptible F344 and resistant Brown Norway (BN) rats. B alleles at Hcs4 locus, on RNO16, control neoplastic nodule volume. We constructed the F344.BN-Hcs4 recombinant congenic strain (RCS) by introgressing a 4.41-cM portion of Hcs4 from BN strain in an isogenic F344 background. Preneoplastic and neoplastic lesions were induced by the "resistant hepatocyte" protocol. Eight weeks after initiation, lesion volume and positivity for proliferating cell nuclear antigen (PCNA) were much higher in lesions of F344 than BN rats of both sexes. These variables were lower in females than in males. Lesion volume and PCNA values of male RCS were similar to those of F344 rats, but in females corresponded to those of BN females. Carcinomatous nodules and HCC developed at 32 and 60 weeks, respectively, in male F344 and congenics and, rarely, in F344 females. BN and congenic females developed only eosinophilic/clear cells nodules. Gonadectomy of congenic males, followed by beta-estradiol administration, caused a decrease in Ar expression, an increase in Er-alpha expression, and development of preneoplastic lesions comparable to those from BN females. Administration of testosterone to gonadectomized females led to Ar increase and development of preneoplastic lesions as in F344 males. This indicates a role of homozygous B alleles at Hcs4 in the determination of phenotypic patterns of female RCS and presence at Hcs4 locus of a high penetrance gene(s), activated by estrogens and inhibited/unaffected by testosterone, conferring resistance to females in which the B alleles provide higher resistance.

Animals↗
PubMed1997

Loss of p21Waf1/Cip1 sensitizes tumors to radiation by an apoptosis-independent mechanism.

Cellular checkpoints are important mediators of the response of normal cells following genotoxic damage, and interruption of these checkpoints is a common feature of many solid tumors. Although the effects of loss in checkpoint function in tumor cells are well understood in terms of cell cycle control, there is little information on their role in determining treatment efficacy in vivo. We have examined both the in vitro and in vivo responses of isogenic lines differing only in the p53-transactivated checkpoint gene, p21Waf1/Cip1. When assayed in vitro, loss of p21 in human colon tumor cells results in a selective induction of apoptosis [Waldman, T., et al., Nature (Lond.), 381: 713-716, 1996.] but no difference in the clonogenic survival. However, when grown as xenografts and irradiated in situ, p21-deficient tumors were significantly more sensitive to radiation as assessed both by clonogenic survival and by regrowth of the tumors following treatment. These data indicate that loss of p21 results in increased sensitivity to killing by ionizing radiation that is independent of the induction of apoptosis and cell cycle arrest but that is specific to cells when they are grown as a solid tumor. These results have important implications for assessing both the genetic determinants of sensitivity to anticancer agents and efficacy of anticancer agents.

Animals↗
PubMed1998

Two distinct phospholipases C of Listeria monocytogenes induce ceramide generation, nuclear factor-kappa B activation, and E-selectin expression in human endothelial cells.

Infection of endothelial cells by Listeria monocytogenes is an essential step in the pathogenesis of listeriosis. We recently reported that L. monocytogenes induces up-regulation of E-selectin and other endothelial adhesion molecules and subsequent polymorphonuclear leukocyte (PMN) adhesion into cultured human endothelial cells. In the present study, we characterized the mechanisms of enhanced E-selectin expression using L. monocytogenes wild type (EGD), the isogenic in-frame deletion mutants for phosphatidylcholine (PC)- and phosphatidylinositol (PI)-specific phospholipases EGD delta plcA and EGD delta plcB, as well as the nonvirulent control strain Listeria innocua. Infection of endothelial cells with EGD delta plcA or EGD delta plcB for 6 h induced, as compared with EGD wild type, intermediate levels of E-selectin mRNA and protein as well as PMN rolling and adhesion at a shear rate of 1 dyne/cm2, indicating that both bacterial phospholipases are required for a maximal effect. Similarly, ceramide content and NF-kappa B activity were increased in L. monocytogenes-exposed endothelial cells, but only to intermediate levels for PC- or PI-phospholipase C (PLC)-deficient listerial mutants. Phospholipase effects could be mimicked by exogenously added ceramides or bacterial sphingomyelinase. The data presented indicate that PI-PLC and PC-PLC are important virulence factors for L. monocytogenes infections that induce accumulation of ceramides that in turn may act as second messengers to control host cell signal-transduction pathways leading to persistent NF-kappa B activation, increased E-selectin expression, and enhanced PMN rolling/adhesion. The ability of L. monocytogenes to stimulate PMN adhesion to endothelial cells may be an important mechanism in the pathogenesis of severe listeriosis.

Cells, Cultured↗
PubMed1993

[The effects of bFGF on the contraction of composite skin grafts].

The authors previously reported that a composite skin graft (CSG) consisting of allodermis graft overlaid with an isogenic epidermis accomplished permanent coverage of a full-thickness skin defect in one operation setting in a rat model. However, the CSG contracted more than the isogenic epidermis alone, presumably due to patchy necrosis of the CSG caused by inadequate blood supply to the isografts. The present study was designed to determine the effects of bFGF on CSG take and its contraction. Rats were divided into two groups. In the tested group the allodermis of CSG was treated with bFGF prior to grafting, while in the control group no treatment was administered. The results showed that the incidence of ischemic necrosis of the isograft was lowered and the area of necrotic patches was lessened in the bFGF group than the control (P < 0.05), and that the contraction of the CSG in the treated group was much less severe than the control as well (P < 0.001). It was found that the extent of necrosis of the CSG was negatively correlated with the resultant area of contraction (r = -0.450, P < 0.05). The study suggests that the ideal results may be obtained when the CSG contracts less than the simple isogenic epidermis graft, provided an optimum treatment modality of CSG by bFGF should be found.

Animals↗
PubMed2000

Streptogramin-based gene regulation systems for mammalian cells.

Here we describe repressible (PipOFF) as well as inducible (PipON) systems for regulated gene expression in mammalian cells, based on the repressor Pip (pristinamycin-induced protein), which is encoded by the streptogramin resistance operon of Streptomyces coelicolor. Expression of genes placed under control of these systems was responsive to clinically approved antibiotics belonging to the streptogramin group (pristinamycin, virginiamycin, and Synercid). The versatility of these systems was demonstrated by streptogramin-regulated expression of mouse erythropoietin (EPO), human placental secreted alkaline phosphatase (SEAP), or green fluorescent protein (GFP) in diverse cell lines (BHK, CHO, HeLa, and mouse myoblasts). Analysis of isogenic constructs in CHO cells demonstrated the PipOFF system gave lower background and higher induction ratios than the widely used tetracycline-repressible (TetOFF) expression systems. The streptogramin-based expression technology was functionally compatible with the TetOFF system, thus enabling the selective use of different antibiotics to independently control two different gene activities in the same cell.

Alkaline Phosphatase↗
PubMed1999

A Target-Specific Cellular Assay for Screening of Topoisomerase I Inhibitors.

We have developed a cellular, target-specific high-throughput assay for the detection of topoisomerase I inhibitors. Topoisomerase I is a nonessential enzyme involved in controlling DNA topology. Topoisomerase I is the target of anticancer drugs such as camptothecin as well as a candidate target for new antifungal drugs. A wild-type Saccharomyces cerevisiae strain and its isogenic topoisomerase I deletion mutant (DeltatopI) were labeled with S65T and wild-type green fluorescent protein (GFP), respectively. We showed that the growth of such a pair of S. cerevisiae strains labeled with this GFP combination can be independently quantified after both strains were mixed. When growth of the mixture of wild-type and DeltatopI strain was monitored in the presence of compounds, only growth of the wild-type strain was inhibited by the topoisomerase I-specific drug camptothecin. In contrast, amphotericin B, a broad-spectrum antifungal drug, inhibited growth of both strains. The two strains were used to screen compound libraries. While 0.9% of all compounds inhibited growth of both strains, only 0.06% inhibited the wild-type but not the DeltatopI strain. Thus, by using a DeltatopI strain as internal control in the same assay mixture, the number of candidate topoisomerase I inhibitors to be retested could be reduced by more than 90%. Further applications of this type of S. cerevisiae-based cellular high-throughput assays will be discussed.

Journal Article↗
PubMed2004

Dual viral transneuronal tracing of central autonomic circuits involved in the innervation of the two kidneys in rat.

The neural control of renal function is exerted by the central nervous system via sympathetic innervation of the kidneys. To determine the extent to which the control of the two kidneys is provided by the same brain neurons, the central circuitry involved in the innervation of both kidneys was characterized in individual rats by dual viral transneuronal tracing using isogenic recombinant strains (PRV-152 and BaBlu) of pseudorabies virus. Prior to dual tracing, the neuroinvasive properties of PRV-152 and BaBlu were characterized by conducting parametric studies, using the two kidneys as an anatomical model, and comparing the pattern of infection with that obtained following injection of the parental strain, PRV-Bartha, into the left kidney. Once the optimal concentrations of virus required to obtain equivalent infection were established, PRV-152 and BaBlu were injected into the left and right kidney, respectively, in the same rats. Immunocytochemical localization of viral reporter proteins at different postinoculation times allowed us to determine the sequence of infection in the brain, as well as to quantify dual- and single-labeled neurons in each infected area. Neurons that influence autonomic outflow to one or both kidneys coexist in all brain areas involved in the control of the sympathetic outflow to the kidneys at every hierarchical level of the circuit. The proportions of dual-infected neurons with respect to the number of total infected neurons varied across regions, but they were maintained at different survival times. The pattern of infection suggests that the activity of each kidney is controlled independently by organ-specific neurons, whereas the functional coordination of the two kidneys results from neurons that collaterize to modulate the sympathetic outflow to both organs. The advantages of using an anatomical symmetrical system, such as the two kidneys, as an experimental approach to characterize PRV recombinants in general are also discussed.

Animals↗
PubMed1997

TraM of plasmid R1 controls transfer gene expression as an integrated control element in a complex regulatory network.

Site-directed mutagenesis was used to investigate the functions of the traM gene in plasmid R1-mediated bacterial conjugation. Three mutant alleles, a null mutation, a sense mutation and a stop mutation, were recombined back into the R1-16 plasmid, a transfer-derepressed (finO-) variant of plasmid R1. The frequency of conjugative transfer of the traM null mutant derivative of R1-16 was 10(7)-fold lower than that of the isogenic parent plasmid, showing the absolute requirement for this gene in conjugative transfer of plasmid R1. Measurements of the abundance of plasmid specified traJ, traA and traM mRNAs, TraM protein levels, and complementation studies indicated that the traM gene of plasmid R1 has at least two functions in conjugation: (i) positive control of transfer gene expression; and (ii) a function in a process distinct from gene expression. Since expression of the negatively autoregulated traM gene is itself affected positively by the expression of the transfer operon genes, this gene constitutes a decisive element within a regulatory circuit that co-ordinates expression of the genes necessary for horizontal DNA transfer. Based on our studies, we present a novel model for the regulation of the transfer genes of plasmid R1 that might also be applicable to other IncF plasmids.

Alleles↗
PubMedDate not supplied

Immune regulation and HLA types in chronic hepatitis.

Studies were undertaken in 32 patients with hepatitis B-negative or -positive chronic active hepatitis or chronic persistent hepatitis to define the relationship between immunoregulatory activity and the HLA-B8 and B12 phenotypes. Suppressor T-cell activity measured by a concanavalin A-induced suppressor system using allogeneic responder cells was dependent on which individual was selected as a source of responder cells. No differences were noted using isogeneic cells as responders. Suppressor T-cell activity measured by the effect of a noninduced suppressor cell on a mixed leukocyte culture was not different from controls. Increased prostaglandin-producing suppressor cell activity was found in patients with hepatitis B-negative (p less than 0.005) and hepatitis B-positive (p less than 0.05) chronic active activity hepatitis. When results of the suppressor activities were compared among patients with chronic hepatitis dependent on the presence of HLA-B8, B12, or neither of these phenotypes, no significant differences were present. These results provide further evidence of altered immunoregulatory function in patients with chronic active hepatitis, which may reflect increased suppression by a population of prostaglandin-producing suppressor cells. The results do not, however, suggest that a gene coding for altered immune regulation is linked to HLA-B8 or B12.

Adult↗
PubMed1986

Hyperglycemia and reproductive defects in non-diabetic gravidas: a mouse model test of a new theory.

The frequent association of various reproductive anomalies with maternal diabetes is explained by a theory that predicts such defects will occur even in transient moderate hyperglycemia in non-diabetic gravidae. A first test of this hypothesis produced strongly supportive results in a mouse model. Thirteen C57BL/6J dams on a grain (control) diet produced (in the 4th week after first mating) 64 pups, 62 surviving to maturity. In the same time interval on a high sucrose diet, fourteen isogenic dams produced only 5 pups, none surviving 5 days (p much less than .001). Additional ongoing studies of the underlying glucose-ascorbate antagonism and of early vs. perinatal hyperglycemia are described briefly.

Animals↗
PubMed2005

Rapamycin treatment is associated with an increased apoptosis rate in experimental vein grafts.

OBJECTIVE: Rapamycin is an immunosuppressive agent with marked antiproliferative properties and is effective in reducing in stent restenosis and vein graft neointimal hyperplasia. Apoptosis is one mechanism counterbalancing cellular proliferation. We therefore investigated the role of apoptosis in rapamycin treated vein grafts in a mouse model. METHODS: C57BL6J mice underwent interposition of the inferior vena cava from isogenic donor mice into the common carotid artery using a cuff technique. In the treatment group 200 microg of rapamycin were applied locally in pluronic gel. The control group did not receive local treatment. Vein grafts were harvested at 4 weeks postoperatively and underwent morphometric analysis as well as immunohistochemical analysis for apoptosis (TUNEL). RESULTS: In grafted veins without treatment (controls) neointimal thickness was 50 (12-58) microm at 4 weeks postoperatively. In 200 microg rapamycin treated grafts the neointimal thickness was 17 (5-55) microm. Rapamycin treated vein grafts showed a significantly increased rate of apoptosis in the adventitia as compared with controls (P=0.032). In the neointima the apoptosis rate was lower in both groups with no significant difference between rapamycin treated grafts and controls. CONCLUSION: We conclude that treatment of experimental vein grafts with rapamycin is associated with an increased apoptosis rate in the vascular wall and a trend towards reduction of neointimal hyperplasia. These results suggest that apoptosis may be a beneficial antiproliferative component for the treatment of vein graft disease.

Animals↗
PubMed2005

Dose-response effects of ectopic agouti protein on iron overload and age-associated aspects of the Avy/a obese mouse phenome.

Isogenic and congenic offspring from matings of inbred black a/a dams by sibling (or non-sibling from another inbred strain) yellow agouti Avy/a sires provide an animal model of obese yellow agouti Avy/a and isogenic lean pseudoagouti Avy/a mice exhibiting two different in vivo concentrations (high, very low) of ectopic agouti protein (ASP) with congenic lean black a/a mice as null controls. This makes it possible to differentiate between the high and very low dose levels of ectopic ASP with respect to interactions with diverse physiological and molecular pathways. Assay of differential responses to 12 or 24 months of carbonyl iron overload assessed the possible suitability of this animal model for the study of hemochromatosis. Agouti A/a B6C3F1 mice were used as non-congenic null controls. The age-related waxing and waning of body weight, food consumption, and caloric efficiency, as well as associated changes in pancreatic islets and islet cells, and formation of liver tumors were assayed. While the hypothesis that these mice might serve as a tool for investigating hemochromatosis was not confirmed, the data did provide evidence that even the very low levels of ASP in pseudoagouti Avy/a mice affect the network of molecular/metabolic/physiological response pathways that comprises the yellow agouti obese phenome. We suggest that the combination of yellow agouti Avy/a, pseudoagouti Avy/a, and black a/a congenic mice provides a practical tool for applying a dose-response systems biology approach to understanding the dysregulatory influence of ectopic ASP on the molecular-physiological matrix of the organism.

Aging↗
PubMedDate not supplied

The antacid Talcid adsorbs and neutralizes all proteins secreted by H. pylori including VacA cytotoxin: a new mechanism for its ulcer-healing action?

BACKGROUND AND AIM: Helicobacter pylori culture supernatant containing VacA cytotoxin significantly inhibits gastric cell proliferation and delays healing of experimental gastric ulcers. Since cell proliferation is crucial for ulcer healing, the removal of inhibitory effects of H. pylori secreted cytotoxin would have a beneficial effect on the healing process. In this study, we determined whether the antacid Talcid can adsorb, remove, or neutralize H. pylori derived VacA cytotoxin responsible for the above deleterious actions. METHODS: Supernatants of viable H. pylori isogenic strains producing VacA cytotoxin [VacA(+)] and with disrupted cytotoxin gene not producing cytotoxin [VacA(-)] were incubated with either placebo, Talcid 10 mg/ml, omeprazole 10 mg/ml (positive control) for 1-24 h. Treated supernatants were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis to evaluate proteins. We also studied the effect of supernatants on epidermal growth factor stimulated Kato III cell proliferation using BrdU labeling. RESULTS: Talcid very effectively removed from the H. pylori culture supernatant the approximately 90 kD VacA(+) cytotoxin at 3 and 24 h (99.5% removal vs. placebo-treated control; p<0.001). It also removed all other proteins, including 66-kD urease and 58-kD heat shock protein, secreted by both VacA(+) and VacA(-) H. pylori strains. Omeprazole was completely ineffective in this regard. Preincubation with Talcid completely abolished the inhibitory effect of VacA(+) H. pylori culture supernatant on epidermal growth factor stimulated Kato III cell proliferation. CONCLUSION: Adsorption and neutralization by Talcid of all H. pylori secreted proteins may explain, at least in part, the ulcer-healing action of this drug.

Aluminum Hydroxide↗
PubMedDate not supplied

The possibility of antiidiotypic activity in multipareous mice.

CBA females pregnant of A/J males were tested for the presence in their sera of antiidiotypic antibodies, capable of combining with antipaternal Ig determinants. Radioimmunoassay tests have shown that in comparison to females pregnant of isogeneic males, allopregnant sera presented values indicating the presence of an antiidiotypic reaction in five out of seven trials. The possible role of antiidiotypic reactions in the control of maternofoetal relationship is discussed.

Animals↗
PubMed1991

Conformational changes induced by integration host factor at origin gamma of R6K and copy number control.

We have investigated the role of integration host factor (IHF) in the replication of plasmid R6K by studying the maintainance of the plasmid in a strain of Escherichia coli that lacks both subunits of IHF and in an isogenic wild type strain and found that all three origins, alpha, beta, and gamma, were functional in the absence of IHF; however, loss of IHF reduced the copy number of those replicons initiating solely from ori gamma by 5-fold. Concomitant loss of direct repeats within the origin that bind the R6K replication initiator protein, Pi, resulted in a further reduction in copy number. Using gel mobility shift analysis, we showed that IHF bound specifically only to one site within the A/T rich region of the minimal origin adjacent to the Pi binding sites. The origin region possessed no intrinsic DNA curvature although IHF induced a strong bend upon binding. Combination footprinting with different orders of addition of Pi and IHF suggested that there was no cooperativity between the two proteins with regard to DNA binding. Hydroxyl-radical footprinting revealed hypersensitive asymmetric periodic cleavage sites within the origin region in the presence of IHF that extended over 200 base pairs and a localized perturbation of cleavage chemistry. The presence of periodic cleavages was dependent upon the presence of the wild type R6K origin sequence and was not observed when the IHF binding site was positioned adjacent to a heterologous sequence. We observed that the conformational changes induced by IHF upon binding to the R6K origin were negatively correlated with the observed decrease in copy number, and therefore, origin conformation altered by protein-DNA interaction may play an important role in the regulation of replication initiation.

Autoradiography↗
PubMed1983

Physiological characterization of Escherichia coli rpoB mutants with abnormal control of ribosome synthesis.

We have previously reported the isolation of Escherichia coli rpoB mutants in which the control of ribosome synthesis by the nucleotide effector guanosine tetraphosphate (ppGpp) is altered, owing to a 20-fold increased sensitivity of the mutant RNA polymerases to ppGpp. In these mutants, the level of ppGpp during exponential growth is decreased about 10-fold, relative to that of rpoB+ wild-type strains, such that a near normal partitioning of RNA polymerase occurs with respect to stable RNA (rRNA and tRNA) gene activity. Here, the physiological effects of two different rpoB alleles in a relA+ and relA background were analyzed in greater detail by comparison with their isogenic rpoB+ wild-type parents. For a given growth medium, the rpoB mutations were found to affect four parameters which resulted in a reduction of growth rate. The results reinforce a previous conclusion that a key element in control of the bacterial growth rate is a mutual relationship between control of ribosome synthesis by ppGpp and control of relA-independent ppGpp metabolism by the concentration and function of ribosomes.

DNA-Directed RNA Polymerases↗
PubMed1985

Evidence for genetic influences on neurotransmitter content of identified neurones of Lymnaea stagnalis.

Neurotransmitter content was measured in two identified giant neurones in isogenic and wild-type populations of the freshwater pond snail Lymnaea stagnalis. The paired serotonergic cerebral giant neurones (LC1 and RC1) have higher transmitter levels and less variability in inbred animals than in wild-type animals. The transmitter content of the unpaired dopaminergic right pedal giant neurone (RPeD1) does not differ between inbred and wild-type animals in either level or variability. It is proposed that serotonin content of the cerebral giant neurones is under partial genetic control, and that animals of the wild-type population may possess a number of different alleles for the genes influencing serotonin levels. Inbreeding resulted in fixation of an allele promoting high serotonin levels. This particular wild-type population is probably already isogenic for genes influencing dopamine content in the right pedal giant neurone.

Animals↗