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At least 451 records · Page 25Linked to original sources
PubMed2005

Oral immunization of mice with lactic acid bacteria producing Helicobacter pylori urease B subunit partially protects against challenge with Helicobacter felis.

BACKGROUND: The development of an efficacious vaccine against infection with Helicobacter pylori, the causative agent of chronic gastritis, peptic ulcer disease, and gastric adenocarcinoma, remains a challenge. Since the use of mucosal adjuvants is limited in human application, we have evaluated the potential of recombinant Lactobacillus strains producing H. pylori urease B (UreB) subunit to deliver this antigen to the gastrointestinal tract. METHODS: Mice were injected orally 3 times with a triple dose of recombinant Lactobacillus plantarum NCIMB8826, the recombinant isogenic cell-wall mutant (alr(-) MD007 strain) expressing UreB, or a mixture of recombinant UreB and cholera toxin (rUreB/CT) as a control. Urease-specific seric immunoglobulin (Ig) G and IgA were measured by use of an enzyme-linked immunosorbent assay. After challenge with Helicobacter felis, stomach infection was examined by use of the rapid urease test and by polymerase chain reaction detection of Helicobacter genomic DNA. RESULTS: Intragastric immunization with both recombinant Lactobacillus strains and rUreB/CT elicited UreB-specific antibodies. After challenge, reduction of H. felis load in the stomachs of mice was observed only after immunization with the recombinant mutant strain MD007 or with rUreB/CT. CONCLUSIONS: This is the first report of successful induction of partial protection against H. felis with a mucosal prime-boost regimen in which recombinant Lactobacillus strains were used as antigen-delivery vehicles.

Adjuvants, Immunologic↗
PubMed1973

Some Physiological Characteristics of the Ethylene-requiring Tomato Mutant Diageotropica.

The diageotropica mutant of tomato (Lycopersicon esculentum Mill.) is shown to require exogenous ethylene for normal growth and development. This single gene mutant is characterized by unsupported horizontal growth of shoots and roots, dark green hyponastic leaf segments, thin rigid stems, and primary and adventitious roots which lack lateral roots. Experiments with growth regulators indicate that the mutant does not produce normal amounts of ethylene in response to auxin treatment. Tests with ethylene-producing compounds or ethylene precursors demonstrate that the mutant requires ethylene for normality. Ethylene concentrations as low as 0.005 microliters per liter are capable of completely normalizing mutant characteristics. This mutant with its isogenic parent variety, cv. VFN8, should be a suitable tool for investigating auxin-stimulated ethylene production and their interrelationship in the control of plant morphology and physiology.

Journal Article↗
PubMed1977

Neuron duplications and deletions in locust clones and clutches.

Duplications and deletions of identified neurons can occur with a high degree of genetic control and specificity, as shown by examining the ocellar interneurons of locusts from different breeding populations, clutches from single mated pairs, and isogenic animals from parthenogenetic clones.

Animals↗
PubMed2004

Identification and characterization of a nontypeable Haemophilus influenzae putative toxin-antitoxin locus.

BACKGROUND: Certain strains of an obligate parasite of the human upper respiratory tract, nontypeable Haemophilus influenzae (NTHi), can cause invasive diseases such as septicemia and meningitis, as well as chronic mucosal infections such as otitis media. To do this, the organism must invade and survive within both epithelial and endothelial cells. We have identified a facilitator of NTHi survival inside human cells, virulence-associated protein D (vapDHi, encoded by gene HI0450). Both vapDHi and a flanking gene, HI0451, exhibit the genetic and physical characteristics of a toxin/antitoxin (TA) locus, with VapDHi serving as the toxin moiety and HI0451 as the antitoxin. We propose the name VapXHi for the HI0451 antitoxin protein. Originally identified on plasmids, TA loci have been found on the chromosomes of a number of bacterial pathogens, and have been implicated in the control of translation during stressful conditions. Translation arrest would enhance survival within human cells and facilitate persistent or chronic mucosal infections. RESULTS: Isogenic mutants in vapDHi were attenuated for survival inside human respiratory epithelial cells (NCI-H292) and human brain microvascular endothelial cells (HBMEC), the in vitro models of mucosal infection and the blood-brain barrier, respectively. Transcomplementation with a vapDHi allele restored wild-type NTHi survival within both cell lines. A PCR survey of 59 H. influenzae strains isolated from various anatomical sites determined the presence of a vapDHiallele in 100% of strains. Two isoforms of the gene were identified in this population; one that was 91 residues in length, and another that was truncated to 45 amino acids due to an in-frame deletion. The truncated allele failed to transcomplement the NTHi vapDHi survival defect in HBMEC. Subunits of full-length VapDHi homodimerized, but subunits of the truncated protein did not. However, truncated protein subunits did interact with full-length subunits, and this interaction resulted in a dominant-negative phenotype. Although Escherichia coli does not contain a homologue of either vapDHi or vapXHi, overexpression of the VapDHi toxin in trans resulted in E. coli cell growth arrest. This arrest could be rescued by providing the VapXHi antitoxin on a compatible plasmid. CONCLUSION: We conclude that vapDHi and vapXHi may constitute a H. influenzae TA locus that functions to enhance NTHi survival within human epithelial and endothelial cells.

Alleles↗
PubMed2005

Inhibition of p53 by lentiviral mediated shRNA abrogates G1 arrest and apoptosis in retinal pigmented epithelial cell line.

We silenced p53 gene expression in ARPE-19, a human retinal pigmented epithelial cell line using RNA interference. The effect of silencing the p53 gene in proliferating ARPE-19 cells was studied. Four short hairpin RNAs (shRNAs) targeting different regions of human p53 mRNA were delivered individually into ARPE-19 cells using lentiviral vector to produce stable cell lines. p53 mRNA and protein levels were reduced to varying extents in the four shRNA-transduced ARPE-19 cell lines. The cell line that showed greatest reduction (85-90%) of p53 expression showed decreased p21 promoter activation after DNA damage with camptothecin, etoposide and MMS. Whereas treatment of wild type ARPE-19 cells with camptothecin resulted in apoptosis, silencing p53 expression increased their survival. Cell cycle analyses indicated that irradiation resulted in a G(1) arrest in ARPE-19 cells, and that the arrest was significantly reduced in p53-silenced cells. Thus, p53 plays a central role in the response of ARPE-19 cells to DNA damaging agents that act via different mechanisms. Additionally, ARPE-19 cells with reduced p53 expression behave similar to tumor cell lines with mutated or non-functional p53. The present data demonstrate the utility of lentiviral vectors to create stable isogenic cell lines with reduced expression of a specific gene, thereby permitting the study of the function of a gene, the pathways controlled by it, and the effect of therapeutics on a cell with altered genetic makeup in a pair-wise fashion.

Apoptosis↗
PubMed1993

Positive and negative regulatory elements control expression of the yeast retrotransposon Ty3.

We report the results of an analysis of Ty3 transcription and identification of Ty3 regions that mediate pheromone and mating-type regulation to coordinate its expression with the yeast life cycle. A set of strains was constructed which was isogenic except for the number of Ty3 elements, which varied from zero to three. Analysis of Ty3 expression in these strains showed that each of the three elements was transcribed and that each element was regulated. Dissection of the long terminal repeat regulatory region by Northern blot analysis of deletion mutants and reporter gene analysis showed that the upstream junction of Ty3 with flanking chromosomal sequences contained a negative control region. A 19-bp fragment (positions 56-74) containing one consensus copy and one 7 of 8-bp match to the pheromone response element (PRE) consensus was sufficient to mediate pheromone induction in either haploid cell type. Deletion of this region, however, did not abolish expression, indicating that other sequences also activate transcription. A 24-bp block immediately downstream of the PRE region contained a sequence similar to the a1-alpha 2 consensus that conferred mating-type control. A single base pair mutation in the region separating the PRE and a1-alpha 2 sequences blocked pheromone induction, but not mating-type control. Thus, the long terminal repeat of Ty3 is a compact, highly regulated, mobile promoter which is responsive to cell type and mating.

Base Sequence↗
PubMed2000

Physiological interactions of phytochromes A, B1 and B2 in the control of development in tomato.

The role of phytochrome B2 (phyB2) in the control of photomorphogenesis in tomato (Solanum lycopersicum L.) has been investigated using recently isolated mutants carrying lesions in the PHYB2 gene. The physiological interactions of phytochrome A (phyA), phytochrome B1 (phyB1) and phyB2 have also been explored, using an isogenic series of all possible mutant combinations and several different phenotypic characteristics. The loss of phyB2 had a negligible effect on the development of white-light-grown wild-type or phyA-deficient plants, but substantially enhanced the elongated pale phenotype of the phyB1 mutant. This redundancy was also seen in the control of de-etiolation under continuous red light (R), where the loss of phyB2 had no detectable effect in the presence of phyB1. Under continuous R, phyA action was largely independent of phyB1 and phyB2 in terms of the control of hypocotyl elongation, but antagonized the effects of phyB1 in the control of anthocyanin synthesis, indicating that photoreceptors may interact differently to control different traits. Irradiance response curves for anthocyanin synthesis revealed that phyB1 and phyB2 together mediate all the detectable response to high-irradiance R, and, surprisingly, that the phyA-dependent low-irradiance component is also strongly reduced in the phyB1 phyB2 double mutant. This is not associated with a reduction in phyA protein content or responsiveness to continuous far-red light (FR), suggesting that phyB1 and phyB2 specifically influence phyA activity under low-irradiance R. Finally, the phyA phyB1 phyB2 triple mutant showed strong residual responsiveness to supplementary daytime FR, indicating that at least one of the two remaining phytochromes plays a significant role in tomato photomorphogenesis.

Base Sequence↗
PubMed1997

Cra-mediated regulation of Escherichia coli adenylate cyclase.

In Escherichia coli, expression of certain genes and operons, including the fructose operon, is controlled by Cra, the pleiotropic catabolite repressor/activator protein formerly known as FruR. In this study we have demonstrated that cra mutant strains synthesize 10-fold less cAMP than isogenic wild-type strains, specifically when grown in fructose-containing minimal media. The glucose-specific IIA protein (IIAglc) of the phosphotransferase system, which activates adenylate cyclase when phosphorylated, is largely dephosphorylated in cra but not wild-type strains growing under these conditions. Dephosphorylation of IIAglc in cra strains apparently results from enhanced fructose operon transcription and fructose uptake. These conclusions were supported by showing that fructose-grown cra strains possess 2.5-fold higher fructose-1-phosphate kinase activity than fructose-grown wild-type strains. Moreover, artificially increasing fructose operon expression in cells transporting fructose dramatically decreased the activity of adenylate cyclase. The results establish that Cra indirectly regulates the activity of adenylate cyclase by controlling the expression of the fructose operon in cells growing with fructose as the sole carbon source.

Adenylyl Cyclases↗
PubMed1988

Regulation of lactate/pyruvate ratios by cyclic AMP in Neurospora crassa.

Cyclic AMP is thought to have a general role in stimulating the breakdown of carbohydrate reserves and subsequent glycolytic activity. This would be expected to increase the availability of reducing equivalents in the form of cytoplasmic NADH. The current study examines another potential reaction controlling cytoplasmic NADH in the fungus Neurospora crassa, that of lactate dehydrogenase, to determine whether it is also regulated by cyclic AMP. The cr-1, adenylate cyclase and cyclic AMP-deficient mutant, grown with and without exogenous cyclic AMP was compared with an isogenic wild type. The results show that cyclic AMP raises pyruvic acid pools and lowers both lactic acid pools and lactate/pyruvate ratios. It does that, in part or in whole, by lowering lactate dehydrogenase activity. The possibility that cytoplasmic NAD+/NADH is a major target of cyclic AMP control is discussed. The high performance liquid chromatography procedures used in these studies are applicable to the measurement of intracellular pools of tricarboxylic acid cycle and other organic acids.

Adenylyl Cyclases↗
PubMed2001

The organization of metabolic reaction networks. III. Application for diauxic growth on glucose and lactose.

A mathematical model to describe carbon catabolite repression in Escherichia coli is developed and in part validated. The model is aggregated from two functional units describing glucose and lactose transport and degradation. Both units are members of the crp modulon and are under control of a global signal transduction system which calculates the signals that turn on or off gene expression for the specific enzymes. Using isogenic mutant strains, our model is validated by a set of experiments. In these experiments, substrate composition of the preculture and of the experimental culture are varied in order to stimulate the system in different ways. With the obtained measurements (three states in the liquid phase and one intracellular component) a part of the model parameters could be estimated. Therefore all experiments could be sufficiently described with a single set of parameters.

Biological Transport↗
PubMed1997

Movement of a small mitochondrial double-stranded RNA element of Cryphonectria parasitica: ascospore inheritance and implications for mitochondrial recombination.

Movement via somatic fusion and inheritance of a small mitochondrial double-stranded (ds) RNA element was examined in Cryphonectria parasitica. The 2.7-kb dsRNA from the C. parasitica strain NB631 encodes a putative RNA-dependent RNA polymerase when the mitochondrial code (UGA = Trp) is invoked. All progeny from asexual spores (conidia) of strain NB631 examined for dsRNA contained the 2.7-kb element. Unlike other C. parasitica dsRNAs, which are cytoplasmic, the dsRNA in strain NB631 was transmitted through the sexual cycle (ascospores) if the strain containing the element acted as the female in crosses. Movement of the 2.7-kb dsRNA was also observed through hyphal anastomosis. Transfer by anastomosis was accompanied by mitochondrial movement and recombination of the mitochondrial genome as determined by RFLP analysis. In control pairings between isolates lacking dsRNA, mitochondrial movement and recombination were also observed. Transfer by anastomosis allowed the generation of infected and uninfected isogenic lines, and permitted us to evaluate the effects of the dsRNA element on virulence of the host. Bark virulence assays on American chestnut suggest that NB631 dsRNA decreases the virulence of C. parasitica, but not to the level associated with members of the Hypoviridae.

Ascomycota↗
PubMed2005

Selective DNA damage responses in murine Xpa-/-, Xpc-/- and Csb-/- keratinocyte cultures.

Cellular DNA damage responses (DDRs) are induced by unrepaired DNA lesions and constitute a protective back-up system that prevents the expansion of damaged cells. These cellular signaling pathways trigger either growth arrest or cell death and are believed to be major components of an early anti-cancer barrier. Cultures of C57BL/6J keratinocytes with various defects in NER sub-pathways allowed us to follow the kinetics of DDRs in an isogenic background and in the proper (physiologically relevant) target cells, supplementing earlier studies in heterogenic human fibroblasts. In a series of well-controlled parallel experiments we have shown that, depending on the NER deficiency, murine keratinocytes elicited highly selective DDRs. After a dose of UV-B that did not affect wild-type keratinocytes, Xpa(-/-) keratinocytes (complete NER deficiency) showed a rapid depletion of DNA replicating S-phase cells, a transient increase in quiescent S-phase cells (not replicating DNA), followed by massive apoptosis. Csb(-/-) keratinocytes (TC-NER deficient) responded by a more sustained increase in QS-phase cells and appeared more resistant to UV-B induced apoptosis than Xpa(-/-). In irradiated Xpc(-/-) keratinocytes (GG-NER deficient) the loss of replicating S-phase cells was associated with a gradual build-up of both QS-phase cells and cells arrested in late-S phase, in complete absence of apoptosis. Our analysis complements and extends previous in vivo investigations and highlights both similarities and differences with earlier fibroblast studies. In vitro cultures of murine keratinocytes provide a new tool to unravel the molecular mechanisms of UV-induced cellular stress responses in great detail and in a physiologically relevant background. This will be essential to fully appreciate the implications of DDRs in tumor suppression and cancer prevention.

Animals↗
PubMed1996

Effects of single P-element insertions on bristle number and viability in Drosophila melanogaster.

Single P-element mutagenesis was used to construct 1094 lines with P[lArB] inserts on all three major chromosomes in an isogenic background previously free of P elements. The effects of insertions on bristle number and on viability were assessed by comparison to 392 control lines. The variance and effects of P-element inserts on bristle number and viability were larger than those inferred from spontaneous mutations. The distributions of effects on bristle number were symmetrical and highly leptokurtic, such that a few inserts with large effects caused most of the increase in variance. The distribution of effects on viability were negatively skewed and platykurtic. On average, the effects of P-element insertions on bristle number were partly recessive and on viability were completely recessive. P-element inserts with large effects on bristle number tended to have reduced viability, but the correlation between the absolute value of the effects on bristle number and on viability was not strong. Fifty P-element inserts tagging quantitative trait loci (QTLs) with large effects on bristle number were mapped cytogenetically. Two P-element-induced scabrous alleles and five extramacrochaetae alleles were generated. Single P-element mutagenesis is a powerful method for identifying QTLs at the level of genetic locus.

Analysis of Variance↗
PubMed2006

Streptococcus pneumoniae enolase is important for plasminogen binding despite low abundance of enolase protein on the bacterial cell surface.

Enolase represents one of the anchorless surface proteins of Streptococcus pneumoniae and has previously been identified as a plasminogen-binding protein, endowing this pathogen with host proteolytic activity. In this study the mAb 245,C-6 (IgG1) was produced in a BALB/c mouse after immunizing with a protein fraction from S. pneumoniae. The mAb reacted with recombinant pneumococcal enolase both under non-denaturing and denaturing conditions. The epitope for the mAb was mapped to residues (55)DKSRYGGLG(63) of pneumococcal enolase using a peptide array. By applying the previously reported structure of enolase, this epitope was localized in a surface-exposed loop in each of the monomers of the octameric enolase. Previous immunoelectron microscopic studies, using polyclonal rabbit antibodies against enolase, depicted enolase on the cell surface but did not quantify the amount of surface-exposed enolase on viable pneumococci. Here, flow cytometry revealed no binding of mAb 245,C-6 to viable pneumococci, including TIGR4 and its non-encapsulated isogenic mutant, and only a minor increase of fluorescence intensity was measured when the polyclonal anti-enolase antibodies were used. In contrast, control antibodies recognizing the choline-binding proteins (CBPs) PspA and PspC showed high reactivities. The non-encapsulated TIGR4 did not show increased levels of antibody binding for mAb 245,C-6 or polyclonal anti-enolase antibodies, but revealed increased binding of polyclonal antibodies reacting with PspA or PspC. These results suggest that, compared to other surface-exposed proteins such as CBPs, the amount of enolase under the selected conditions is low. Flow cytometry, however, with FITC-labelled plasminogen demonstrated that the amount of surface-exposed enolase is important for plasminogen binding and, therefore, is also important for pneumococcal pathogenesis.

Antibodies, Bacterial↗
PubMed2000

Functional analysis of heterologous holin proteins in a lambdaDeltaS genetic background.

Holins are small hydrophobic proteins causing non-specific membrane lesions at the end of bacteriophage multiplication, to promote access of the murein hydrolase to their substrate. We have established a lambdaDeltaS genetic system, which enables functional expression of holins from various phages in an isogenic phage lambda background, and allows qualitative evaluation of their ability to support lysis of Escherichia coli cells. Synthesis of Holins is under control of native lambda transcription and translation initiation signals, and the temperature-sensitive CIts857 repressor. A number of different holins were tested in this study. The opposing action of phage lambda S105 and S107 holin variants in lysis timing could be confirmed, whereas we found evidence for a functionally non-homologous dual translational start motif in the Listeria phage Hol500 holin, i.e., the Hol500-96 polypeptide starting at Met-1 revealed a more distinct lytic activity as compared to the shorter product Hol500-93. The largest holin known, HolTW from a Staphylococcus aureus phage, revealed an early lysis phenotype in the lambdaDeltaSthf background, which conferred a plaque forming defect due to premature lysis. Mutant analysis revealed that an altered C-terminus and/or a V52L substitution were sufficient to delay lysis and enable plaque formation. These results suggest that the extensively charged HolTW C-terminus may be important in regulation of lysis timing. The gene 17.5 product of E. coli phage T7 was found to support sudden, saltatory cell lysis in the lambdaDeltaSthf background, which clearly confirms its holin character. In conclusion, lambdaDeltaSthf offers a useful genetic tool for studying the structure-function relationship of the extremely heterogeneous group of holin protein orthologs.

Amino Acid Sequence↗
PubMed2004

Ataxia-telangiectasia mutated gene controls insulin-like growth factor I receptor gene expression in a deoxyribonucleic acid damage response pathway via mechanisms involving zinc-finger transcription factors Sp1 and WT1.

The IGF-I receptor (IGF-IR) has a central role in cell cycle progression as well as in the establishment of the transformed phenotype. Increased expression of the IGF-IR gene, in addition, is correlated with acquisition of radioresistance for cell killing. The ataxia-telangiectasia mutated (ATM) gene product has a pivotal role in coordinating the cellular response to DNA damage. The present study was aimed at testing the hypothesis that the ability of ATM to coordinate the DNA damage response that will lead to cell survival or, alternatively, to apoptosis depends, to a significant extent, on its capacity to control IGF-IR gene expression. The potential involvement of ATM in regulation of IGF-IR expression and function was investigated in isogenic cells with and without ATM function [AT22IJE-T/pEBS7 (ATM -/-) and ATM-corrected AT22IJE-T/YZ5 (ATM +/+) cells and 293 human embryonic kidney cells transfected with small interfering RNAs targeted to ATM]. In addition, the effect of ATM on IGF-IR expression was assessed in nonisogenic cells with ATM function (HFF + human telomerase reverse transcriptase) and without ATM function (GM5823 + human telomerase reverse transcriptase). Results obtained showed that IGF-IR gene expression and IGF-IR promoter activity were largely reduced in ATM -/- cells. Addition of the radiomimetic agent neocarzinostatin for 4 h, however, induced a significant increase in IGF-IR levels in cells without ATM function. In addition, IGF-I-induced IGF-IR and insulin receptor substrate-1 phosphorylation were greatly impaired in ATM-deficient cells. Furthermore, we identified zinc-finger transcription factors Sp1 and WT1 as potential mediators of the effect of ATM on IGF-IR gene expression. The present data suggests that the IGF-IR gene is a novel downstream target in an ATM-mediated DNA damage response pathway. Deregulated expression of the IGF-IR gene after ionizing radiation may be linked to genomic instability and enhanced transforming capacity.

Ataxia Telangiectasia Mutated Proteins↗
PubMed1985

Characteristics of spontaneously hypertensive, Wistar Kyoto and Munich Wistar rats bred in Brazil.

The breeding of imported strains of isogenic rats was started in 1981 because of the lack of experimental rat models in Brazil. The imported strains were: Spontaneously Hypertensive Rats (SHR) and their normotensive controls Wistar Kyoto Rats (WKY) from the National Institutes of Health, Bethesda, MD, and Munich Wistar (MW) rats which present superficial renal glomeruli, from Simonsen Laboratories, Gilroy, CA. Breeding of these strains was carried out without strict barriers (conventional breeding), under Standard Operating Procedures and strict inbreeding. Environmental factors such as ration, light, temperature, type of shavings and bedding, size of cages and their population were constant. Body weight growth curves were constructed for the three strains. The productivity of imported rats and local breeding colonies in 1981, 1982 and 1983 was compared on the basis of the following parameters: mean litter size, productivity of the females, pre- and post-weaning mortality, and effective yield. Systolic blood pressure was also measured for SHR and WKY rats. MW rats showed a high and relatively stable reproduction performance. The productivity of SHR and especially WKY animals declined progressively during the first three years, making the breeding of these strains of isogenic rats very difficult.

Animals↗
PubMed1982

Control of RNA synthesis in Escherichia coli after a shift to higher temperature.

Parameters of RNA synthesis were measured after a temperature upshift in a pair of Escherichia coli B/r strains that are isogenic except for having relA and relA+ loci, to examine the cause for a reported anomaly in the correlation between guanosine tetraphosphate (ppGpp) and stable RNA (rRNA, tRNA) synthesis under such conditions. Two main results were: (i) the specific stable RNA gene activity (stable RNA per total RNA synthesis) correlated in the conventionally expected fashion with the level of ppGpp but was obscured by a nonspecific increase in the RNA chain elongation rate due to the higher temperature; (ii) the temperature upshift caused a transient reduction in the RNA polymerase activity (transcribing per total enzyme) that accounts for the previously observed oscillating RNA synthesis rate after a temperature shift.

Bacterial Proteins↗