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PubMed1997

Graft damage after a single lung transplantation for pulmonary hypertension in a rat model.

The hemodynamic effect and degree of damage in grafts of single lung transplants for pulmonary hypertension were studied in rats with monocrotaline-induced pulmonary hypertension. Inbred male Lewis rats (weight 200-230 g) were divided into two groups. Group 1 (control group, n = 16) underwent isogenic left lung transplantation, while group 2 (n = 15) received an intravenous administration of monocrotaline (80 mg/kg i.v.) and underwent isogenic left single lung transplantation 3 week later. Hemodynamic evaluations were performed prior to transplantation, at 1 h postoperatively, and on days 3 and 7 after transplantation. Mean pulmonary arterial pressure (mPAP) rapidly declined after transplantation in group 2, from 39.3 +/- 8.7 mmHg to 18.5 +/- 3.0 mmHg 1 h after transplantation, and remained stable on day 7 after transplantation. No significant difference in the mPAP between the two groups was observed after transplantation. The extravascular lung water volume (ELWV: dry/wet ratio) in the right lung of group 2 significantly increased on day 3 (0.86 +/- 0.02) (P < 0.01), and subsequently decreased to control levels on day 7 (0.83 +/- 0.02). There was no significant difference in the ELWV in the grafted lungs between the two groups (0.84 +/- 0.03 vs 0.86 +/- 0.04), but there was tendency toward an increase in ELWV in group 2 on days 3 and 7. These data thus demonstrated that a hemodynamic improvement was obtained by single lung transplantation; however the degree of graft damage was remarkable in the pulmonary hypertension group.

Animals↗
PubMed1995

Control of glucose influx into glycolysis and pleiotropic effects studied in different isogenic sets of Saccharomyces cerevisiae mutants in trehalose biosynthesis.

The GGS1/TPS1 gene of the yeast Saccharomyces cerevisiae encodes the trehalose-6-phosphate synthase subunit of the trehalose synthase complex. Mutants defective in GGS1/TPS1 have been isolated repeatedly and they showed variable pleiotropic phenotypes, in particular with respect to trehalose content, ability to grow on fermentable sugars, glucose-induced signaling and sporulation capacity. We have introduced the fdp1, cif1, byp1 and glc6 alleles and the ggs1/tps1 deletion into three different wild-type strains, M5, SP1 and W303-1A. This set of strains will aid further studies on the molecular basis of the complex pleiotropic phenotypes of ggs1/tps1 mutants. The phenotypes conferred by specific alleles were clearly dependent on the genetic background and also differed for some of the alleles. Our results show that the lethality caused by single gene deletion in one genetic background can become undetectable in another background. The sporulation defect of ggs1/tps1 diploids was neither due to a deficiency in G1 arrest, nor to the inability to accumulate trehalose. Ggs1/tps1 delta mutants were very sensitive to glucose and fructose, even in the presence of a 100-fold higher galactose concentration. Fifty-percent inhibition occurred at concentrations similar to the Km values of glucose and fructose transport. The inhibitory effect of glucose in the presence of a large excess of galactose argues against an overactive glycolytic flux as the cause of the growth defect. Deletion of genes of the glucose carrier family shifted the 50% growth inhibition to higher sugar concentrations. This finding allows for a novel approach to estimate the relevance of the many putative glucose carrier genes in S. cerevisiae. We also show that the GGS1/TPS1 gene product is not only required for the transition from respirative to fermentative metabolism but continuously during logarithmic growth on glucose, in spite of the absence of trehalose under such conditions.

Cell Division↗
PubMed1996

Delay of the denervation process in skeletal muscle by sensory ganglion graft and its clinical application.

We examined the effects of dorsal root ganglion isografts on the denervation process of skeletal muscle. A segment of sciatic nerve was removed from each of 25 inbred Wistar-Kyoto rats. Fifteen were set aside as controls. In the remaining 10 rats, isogeneic cervical dorsal root ganglia were grafted to the severed distal stump of the common peroneal nerve. Between day 72 and day 286 postoperatively, both controls and recipients were killed after twitch and tetanic tension recording of the extensor digitorum longus was performed. The wet muscle weight and the twitch and tetanic tensions of the denervated extensor digitorum longus in the graft group were significantly greater than those in the control group. The mean area of the denervated tibialis anterior muscle fibers in the graft group also was significantly larger than that in the control group. In electron and light microscopic images, nerve cells along the periphery of each dorsal root ganglion were found surviving with regenerating axons throughout the experimental period. Numerous myelinated axons were observed in the common peroneal nerve of the graft group, and there were significantly more axonal branches in the extensor digitorum longus of the graft group than in the extensor digitorum longus of the control group. Thus sensory nerve fibers from the grafted dorsal root ganglia had certain beneficial effects to slow the denervation process, presumably secreting trophic factors into the denervated muscle. Clinically, we have transferred avulsed dorsal root ganglia in cases of total brachial plexus avulsion directly into denervated skeletal muscle. This procedure, accompanied by nerve crossing procedures, will probably keep denervated skeletal muscle in a better condition until regenerating motor axons from the repair site reach their target muscle.

Animals↗
PubMed1998

Use of a genetically enhanced, pediocin-producing starter culture, lactococcus lactis subsp. lactis MM217, To control listeria monocytogenes in cheddar cheese

Cheddar cheese was prepared with Lactococcus lactis subsp. lactis MM217, a starter culture which contains pMC117 coding for pediocin PA-1. About 75 liters of pasteurized milk (containing ca. 3.6% fat) was inoculated with strain MM217 (ca. 10(6) CFU per ml) and a mixture of three Listeria monocytogenes strains (ca. 10(3) CFU per ml). The viability of the pathogen and the activity of pediocin in the cheese were monitored at appropriate intervals throughout the manufacturing process and during ripening at 8 degreesC for 6 months. In control cheese made with the isogenic, non-pediocin-producing starter culture L. lactis subsp. lactis MM210, the counts of the pathogen increased to about 10(7) CFU per g after 2 weeks of ripening and then gradually decreased to about 10(3) CFU per g after 6 months. In the experimental cheese made with strain MM217, the counts of L. monocytogenes decreased to 10(2) CFU per g within 1 week of ripening and then decreased to about 10 CFU per g within 3 months. The average titer of pediocin in the experimental cheese decreased from approximately 64,000 arbitrary units (AU) per g after 1 day to 2,000 AU per g after 6 months. No pediocin activity (<200 AU per g) was detected in the control cheese. Also, the presence of pMC117 in strain MM217 did not alter the cheese-making quality of the starter culture, as the rates of acid production, the pH values, and the levels of moisture, NaCl, and fat of the control cheese and the experimental cheese were similar. Our data revealed that pediocin-producing starter cultures have significant potential for protecting natural cheese against L. monocytogenes.

Journal Article↗
PubMed1999

Fimbria-mediated enhanced attachment of nontypeable Haemophilus influenzae to respiratory syncytial virus-infected respiratory epithelial cells.

Respiratory syncytial virus (RSV) infection is known to predispose children to otitis media and sinusitis due to bacteria such as nontypeable Haemophilus influenzae (NTHI). In this study, we investigated the role of NTHI surface outer membrane protein P5-homologous fimbriae (P5-fimbriae) in attachment to RSV-exposed A549 epithelial cells. Analysis by fluorescence flow cytometry showed that a live P5-fimbriated NTHI strain (NTHIF+) attached to a higher proportion of RSV-exposed A549 cells than to control cells (mean, 68% for RSV versus 29% for control; P = 0.008), while attachment of the P5-fimbriae-deficient isogenic mutant strain (NTHIF-) was significantly lower than in control cells and rose only slightly following RSV exposure (mean, 17% for RSV versus 10% for control, P = 0.229). Attachment of NTHIF+ did not correlate with the amount of RSV antigen expressed by A549 cells. Furthermore, paraformaldehyde-fixed NTHIF+ also demonstrated an enhanced binding to RSV-exposed cells. Observations by transmission electronic microscopy showed that the mean number of bacteria attached per 100 RSV-exposed A549 cells was higher for NTHIF+ than NTHIF- (99 versus 18; P < 0.001). No intracellular bacteria were identified. UV-irradiated conditioned supernatants collected from RSV-infected A549 cultures (UV-cRSV) also enhanced the attachment of NTHIF+ to A549, suggesting the presence of a preformed soluble mediator(s) in UV-cRSV that enhances the expression of receptors for P5-fimbriae on A549 cells. In summary, RSV infection significantly enhances NTHI attachment to respiratory epithelial cells. P5-fimbria is the critical appendage of NTHI that participates in this attachment. In clinical settings, blocking of the P5-fimbria-mediated attachment of NTHIF+ by passive or active immunity may reduce the morbidity due to NTHI during RSV infection.

Antigens, Viral↗
PubMed2000

Fumarate reductase is essential for Helicobacter pylori colonization of the mouse stomach.

Fumarate reductase (FRD) is the key enzyme in fumarate respiration induced by anaerobic growth of bacteria. In Helicobacter pylori, this enzyme appears to be constitutively expressed under microaerobic conditions and is not essential for its survival in vitro. In this study, the role of FRD in the colonization of H. pylori was investigated using a mouse model. The frdA gene coding for subunit A of FRD, and two control genes, copA and copP associated with the export of copper out of H. pylori, were inactivated by insertion of the chloramphenicol acetyltransferase cassette into these individual genes. The isogenic mutants of H. pylori strain AH244 were obtained by natural transformation. Seventy-five ICR mice (15 mice/group) were orogastrically dosed with either the wild type H. pylori strain AH244, its isogenic mutants, or Brucella broth (negative control). Five mice from each group were killed at 2, 4 and 8 weeks post-inoculation (WPI), respectively. H. pylori colonization was not detected in mouse gastric mucosa infected with the frdA mutant at any time point in the study by both quantitative culture and PCR. In contrast, the mice inoculated with either wild type AH244, copA or copPH. pylori mutants became readily infected. These data indicate that FRD plays a crucial role in H. pylori survival in the gastric mucosa of mice. Given that FRD, present in all H. pylori strains, is immunogenic in H. pylori -infected patients and H. pylori growth in vitro can be inhibited by three anthelmintics (morantel, oxantel and thiabendazole), this enzyme could potentially be used both as a novel drug target as well as in the development of vaccines for H. pylori prevention and eradication.

Animals↗
PubMed2006

Combination tetrathiomolybdate and radiation therapy in a mouse model of head and neck squamous cell carcinoma.

OBJECTIVE: To assess the effect of combining tetrathiomolybdate therapy and radiation treatment (RT) on tumor growth in the mouse head and neck squamous cell carcinoma (HNSCC) model. DESIGN: One million HNSCC cells were injected subcutaneously into the flanks of C3H/HeJ mice and the tumors grown to an average of 301 mm3 (day 0). Mice were randomized into 4 groups: (a) no therapy, (b) tetrathiomolybdate alone, (c) RT alone, or (d) tetrathiomolybdate + RT. Data from 3 experiments with these 4 groups were analyzed. A gaussian mixed model was fit to the initialized logarithm of the tumor size counts between days 7 and 16 (linear component), and growth rates were compared. Assays using 3-(4,5-dimethylthiazol-2yl)-2,5 diphenyltetrazolium bromide (MTT) were conducted on HNSCC cells in culture with varying doses of tetrathiomolybdate. INTERVENTIONS: Treated mice were given tetrathiomolybdate in their water and observed for clinical evidence of toxic effects associated with copper depletion as measured by ceruloplasmin assay. When tumor sizes reached an average of 535 mm(3), mice receiving RT were given a single fraction of 750 rad (7.5 Gy), a dose determined in previous experiments to slow but not cure tumor growth, permitting an examination of interaction of radiation with tetrathiomolybdate. RESULTS: Data from 3 separate experiments were analyzed. There were a total of 37 mice in the untreated group, 32 mice in the tetrathiomolybdate alone group, 38 mice in the RT alone group, and 46 mice in the tetrathiomolybdate + RT group. Ceruloplasmin assays showed that we had obtained adequate copper reduction throughout the experiments to inhibit angiogenesis with minimal toxic effects. The tetrathiomolybdate + RT combined therapy group of mice showed a statistically significant decrease in tumor growth compared with both the tetrathiomolybdate alone (P = .001) and RT alone groups (P<.001). CONCLUSION: The combination of the anti-angiogenic copper chelating agent tetrathiomolybdate with RT improved local control of HNSCC in an isogenic mouse model compared with either therapy alone.

Angiogenesis Inhibitors↗
PubMed1998

Role of fimbriae-mediated adherence for neutrophil migration across Escherichia coli-infected epithelial cell layers.

This study examined the role of P and type 1 fimbriae for neutrophil migration across Escherichia coli-infected uroepithelial cell layers in vitro and for neutrophil recruitment to the urinary tract in vivo. Recombinant E. coli K-12 strains differing in P or type 1 fimbrial expression were used to infect confluent epithelial layers on the underside of transwell inserts. Neutrophils were added to the upper well, and their passage across the epithelial cell layers was quantified. Infection with the P- and type 1-fimbriated recombinant E. coli strains stimulated neutrophil migration to the same extent as a fully virulent clinical E. coli isolate, but the isogenic non-fimbriated vector control strains had no stimulatory effect. The enhancement of neutrophil migration was adhesion dependent; it was inhibited by soluble receptor analogues blocking the binding of P fimbriae to the globoseries of glycosphingolipids or of type 1 fimbriae to mannosylated glycoprotein receptors. P- and type 1-fimbriated E. coli triggered higher interleukin (IL) 8 secretion and expression of functional IL-8 receptors than non-fimbriated controls, and the increase in neutrophil migration across infected cell layers was inhibited by anti-IL-8 antibodies. In a mouse infection model, P- or type 1-fimbriated E. coli stimulated higher chemokine (MIP-2) and neutrophil responses than the non-fimbriated vector controls. The results demonstrated that transformation with the pap or fim DNA sequences is sufficient to convert an E. coli K-12 strain to a host response inducer, and that fimbriation enhances neutrophil recruitment in vitro and in vivo. Epithelial chemokine production provides a molecular link between the fimbriated bacteria that adhere to epithelial cells and tissue inflammation.

Animals↗
PubMed1988

The effect of sequence homozygosity on the frequency of X-chromosomal exchange in Drosophila melanogaster females.

The repair of mismatched heteroduplex DNA has been implicated in the normal resolution of meiotic exchange events. Although sequence microheterogeneity over defined intervals of homologous chromosomes has been correlated with local effects on recombination, this correlation has not previously been extended to effects on chromosomal levels of exchange. In order to determine the role of microheterogeneity in normal exchange between homologs, a system was devised for monitoring exchange between isogenic X chromosomes. Lack of microheterogeneity did not significantly alter the frequency of exchange along the isogenic X chromosomes relative to controls or to previously reported values. There were, however, characteristic levels of exchange intrinsic to the cloned X chromosomes in each of the lines tested.

Animals↗
PubMed1994

Loss of function mutation in the yeast multiple drug resistance gene PDR5 causes a reduction in chloramphenicol efflux.

The yeast (Saccharomyces cerevisiae) PDR5 gene product encodes a 160-kDa protein related to the large ABC family of transporters, including the human MDR1 multidrug resistance p-glycoprotein. Loss of function mutations in PDR5 result in chloramphenicol hypersensitivity. A pdr5::Tn5 loss of function mutant exhibits a markedly impaired efflux of chloramphenicol compared with that of an isogenic PDR5 (wild-type) control.

ATP-Binding Cassette Transporters↗
PubMed2004

Helicobacter pylori induces apoptosis of macrophages in association with alterations in the mitochondrial pathway.

Helicobacter pylori is a gastric bacterial pathogen that evades host immune responses in vivo and is associated with the development of gastritis, peptic ulcer disease, and gastric cancers. Induction of macrophage apoptosis is a method employed by multiple pathogens to escape host immune responses. Therefore, we hypothesized that H. pylori induces apoptosis of infected macrophages. RAW 264.7 cells were infected with H. pylori strain 60190, and apoptosis was assessed. Transmission electron microscopy and fluorescence microscopy showed that infected macrophages displayed morphological features characteristic of apoptosis. Quantification by acridine orange-ethidium bromide fluorescent-dye staining showed that apoptosis was dose and time dependent, and apoptosis was further confirmed by increased binding of annexin V-fluorescein isothiocyanate (FITC) to externalized phosphatidylserine of infected but not of control macrophages. Macrophages infected with isogenic mutants of H. pylori strain 60190 deficient in either cagA or vacA induced significantly less apoptosis than the parental strain, as assessed by increased binding of annexin V-FITC. Western blot analysis of whole-cell protein lysates revealed that infection with strain 60190 induced a time-dependent increase in cleavage of procaspase 8 and disappearance of full-length Bid compared with uninfected cells. Furthermore, pharmacological inhibition of caspase 8 caused a decrease in levels of apoptosis. Finally, infection caused a time-dependent increase in mitochondrial-membrane permeability and release of cytochrome c into the cytosol. These results suggest that H. pylori induces apoptosis of macrophages in association with alterations in the mitochondrial pathway. Elimination of this key immunomodulatory cell may represent a mechanism employed by the bacterium to evade host immune responses.

Animals↗
PubMed1988

Mutual antagonism among killer yeasts: competition between K1 and K2 killers and a novel cDNA-based K1-K2 killer strain of Saccharomyces cerevisiae.

Mutually antagonistic K1 and K2 killer strains compete when mixed and serially subcultured. At pH 4.6, where the K1 killer toxin is more stable in vitro, the K1 strain outcompeted the K2 strains at both 18 and 30 degrees C. At pH 4.0, closer to the in vitro pH optimum of the K2 killer toxin, the K1 strain again predominated at 18 degrees C, but at 30 degrees C the K2 strains became the sole cell type on subculture. To show more clearly that these results were dependent upon the respective killer toxins, control experiments were conducted with isogenic, nonkiller strains cured of the dsRNA-based killer virions. Such nonkiller strains were unable to compete with antagonistic killers under conditions where their isogenic killer parents could, strongly suggesting that the killer phenotype was important in these competitions. Double K1-K2 killer strains cannot stably exist, as their dsRNA genomes compete at a replicative level. Using recombinant DNA methodology, a stable K1-K2 killer strain was constructed. This strain outcompeted both K1 and K2 killers when serially subcultured under conditions where either the K1 or the K2 strains would normally predominate in mixed cultures. Such a double killer may be useful in commercial fermentations, where there is a risk of contamination by killer yeasts.

DNA↗
PubMed1992

Cross-linking of the gamma subunit of the Escherichia coli ATPase (ECF1) via cysteines introduced by site-directed mutagenesis.

The gamma subunit of the Escherichia coli F1 ATPase (ECF1) has been altered by site-directed mutagenesis to create five different mutants, gamma-S8C, gamma-S81C, gamma-T106C, gamma-S179C, and gamma-V286C, respectively. ECF1 isolated from four of these mutants had ATPase activities similar to that of a wild-type isogenic strain used as a control, the exception was enzyme isolated from mutant gamma-S81C, which had an ATPase activity of around 70-80% of the wild type. ECF1 isolated from each of the various mutants was reacted with N-(4-(7-(diethylamino)-4-methylcoumarin-3-yl))maleimide (CM). The fluorescent reagent was incorporated into Cys residues placed at positions 8, 106, 179, and 286, but not at 81, indicating which of these Cys residues are on the surface of the gamma subunit in the enzyme complex. Modification of the Cys at position 106 with CM activated the enzyme, and modification of the Cys at position 8 inhibited ATPase activity a small amount; however, modification of Cys at 179 or 286 had no effect on enzyme activity. The four mutants with a reactive Cys were reacted with tetrafluorophenylazide maleimides (TFPAMs), novel photoactivatable cross-linkers. In the mutant gamma-S8C, cross-links were formed between the introduced Cys on the gamma subunit and sites on the beta subunit. This cross-linking between gamma and beta depended on nucleotide conditions under which the photolysis was carried out, with differently migrating cross-linked products being obtained in ATP + EDTA compared with ATP + Mg2+ or ATP + Mg2+ Pi. Cross-linking between beta and gamma inhibited ATPase activity in proportion to the yield of cross-linked product. In the mutant gamma-V286C, cross-links were formed between the introduced Cys on gamma and the alpha subunit which were the same in all nucleotide conditions and which led to inhibition of ATPase activity.

Adenosine Triphosphatases↗
PubMed1991

Fecal fat, cyclosporine, and alpha 1-antitrypsin for assessment of small bowel function following transplantation.

Many factors affect the integrity of transplanted small bowel. These include ischemic preservation and immunologic injury as well as the division of intestinal lymphatics during transplantation. This study was undertaken to evaluate the recovery of fat absorption in transplanted small bowel in syngeneic rats. Orthotopic transplantation of the total small bowel with resection of the native intestine was performed. The experimental (n = 11) and a pair-fed, sham-operated control (n = 8) groups were fed a 50% kcal corn oil semipurified diet. Studies of cyclosporine (CSA) absorption, maltose absorption, dietary fat, and fecal alpha 1-antitrypsin (FA1AT) excretion in transplanted animals were performed preoperatively and at 15, 30, and 50 days postoperatively. There was no significant difference in the weight change or fat and maltose absorption in experimental animals compared with control animals at any time point. Peak serum CSA levels were lower at day 15 in transplanted animals than in controls (P = 0.006) and improved but remained lower than those in controls at days 30 and 50 (P = 0.017). FA1AT excretion was increased on postoperative day 15 (accompanied by a decrease in body weight) and returned to control levels at days 30 and 50. Transplanted isogeneic rats had weight recovery and fat and carbohydrate absorption similar to those of controls. Transplanted animals had a protein-losing enteropathy measured by FA1AT at day 15 that resolved by 30 and 50 days, respectively. CSA absorption showed a much more gradual return to control levels and remained abnormal at 50 days.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1998

ATP-dependent transport of reduced glutathione on YCF1, the yeast orthologue of mammalian multidrug resistance associated proteins.

The transport systems involved in the export of cellular reduced glutathione (GSH) have not been identified, although recent studies implicate a role for some of the multidrug resistance associated proteins (MRP), including MRP1 and MRP2. The present study examined the hypothesis that the yeast orthologue of MRP, Ycf1p, mediates ATP-dependent GSH transport. [3H]GSH transport was measured in vacuolar membrane vesicles isolated from a control strain of Saccharomyces cerevisiae (DTY165), the isogenic DTY167 strain that lacks a functional Ycf1p, and in DTY167 transformed with a 2-micrometer plasmid vector containing YCF1. GSH transport in control vacuolar membrane vesicles was mediated largely by an ATP-dependent, low affinity pathway (Km = 15 +/- 4 mM). ATP-dependent [3H]GSH transport was cis-inhibited by substrates of the yeast Ycf1p transporter and inhibited by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, probenecid, and sulfinpyrazone, inhibitors of MRP1 and MRP2, but was minimally affected by membrane potential or pH gradient uncouplers. In contrast, ATP-dependent GSH transport was not seen in vacuolar membrane vesicles isolated from the DTY167 yeast strain without a functional Ycf1p but was restored to near wild-type levels in the DTY167 strain transformed with YCF1 and expressing the vacuolar Ycf1p transporter. On the other hand, expression and functional activity of a bile acid transporter, Bat1p, and of the V-type ATPase were similar in all three yeast strains. These results provide direct evidence for ATP-dependent low affinity transport of GSH by the yeast Ycf1p transporter. Because of the structural and functional homology between Ycf1p and MRP1 and MRP2, these data support the hypothesis that GSH efflux from mammalian cells is mediated by these membrane proteins.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

Maternal, Single-Gene Regulation of Assimilate Partitioning in Pea.

Assimilate partitioning has been identified as a key process in the control of yield. Although the role of reproductive structures in this process has received intensive study, our understanding of the role of the maternal plant is limited. We suggest that the Sn gene of pea (Pisum sativum L.) is a potentially valuable genetic tool for studying maternal regulation of partitioning. In this study, nearly isogenic lines differing at the Sn locus were compared with respect to seed-filling characteristics and carbon assimilation. Lines with the Sn gene had a slower rate and shorter duration of seed growth than the line recessive for this gene, and these traits could not be ascribed to reduced carbon assimilation. Flowers of the two nearly isogenic lines were manually pollinated to control the genotype of the developing embryo independently of the maternal genotype. The final dry weight of the seed was determined by the genotype of the maternal plant and not by the genotype of the embryo, supporting the hypothesis that the Sn gene acts in the vegetative plant to regulate the partitioning of assimilates between vegetative and reproductive growth. Although the Sn gene has been noted for delaying apical senescence, it also delayed leaf senescence in this study; leaves of the Sn line continued to photosynthesize long past the time that leaves of the recessive line had senesced and after the seeds and pods were dry.

Journal Article↗
PubMed1983

rpoB mutation in Escherichia coli alters control of ribosome synthesis by guanosine tetraphosphate.

An isogenic pair of relA+ and relA strains of Escherichia coli B/r with a mutation in the RNA polymerase subunit gene rpoB (Rifr) was isolated in which the relationship between guanosine tetraphosphate (ppGpp) concentration and stable RNA (rRNA, tRNA) gene activity was altered. The RNA polymerase in the rpoB strains was found to be about 20-fold more sensitive to ppGpp with respect to its stable RNA promoter activity than was the wild-type enzyme. The existence of such mutants is consistent with the idea that ppGpp interacts with the RNA polymerase enzyme and thereby alters its promoter selectivity, i.e., reduces its affinity for the stable RNA promoters. Under most conditions, the rpoB mutants had a reduced rate of growth and about a 10-fold-reduced intracellular concentration of ppGpp compared with the rpoB wild-type strains. The reduction of the level of ppGpp in the rpoB mutants during exponential growth was presumably a reflection of an indirect effect of the rpoB mutation on the control of relA-independent ppGpp metabolism.

DNA-Directed RNA Polymerases↗
PubMed2001

Photosynthetic control of the plasma membrane H+-ATPase in Vallisneria leaves. II. Presence of putative isogenes and a protein equipped with a C-terminal autoinhibitory domain.

In vitro treatment with trypsin of plasma membrane (PM) vesicles isolated from the leaves of Vallisneria gigantea Graebner, an aquatic monocot, produced a marked decrease in the Km for ATP and an increase in the Vmax of H+-transporting activity. Concomitantly, the removal of 8 kDa of the C-terminal domain from the 94-kDa PM H+-ATPase was confirmed by immunoblotting using different kinds of polyclonal antibody. Three partial clones of putative PM H+-ATPase genes (Vga1, 2, and 3) were isolated from leaves by reverse transcription polymerase chain reaction. Northern blotting analysis revealed that the expression level of Vga3 was high and that of the other two genes was much lower. The H+-transporting activity of PM vesicles was substantially suppressed in the presence of inorganic phosphate (Pi), which has been supposed to be a noncompetitive inhibitor of the PM H+-ATPase, coincident with an increase in the Km for ATP and a decrease in the Vmax. After treatment of the isolated PM vesicles with trypsin, the inhibitory effect of Pi was no longer evident. This result indicates that Pi inhibited the activity through the C-terminal autoinhibitory domain of the PM H+-ATPase. Furthermore, Pi increased the Km for ATP of the H+-transporting activity in the PM vesicles isolated from both dark-adapted and red-light-irradiated leaves. The results suggest that regulation of the Km for ATP through the operation of photosynthesis is independent of regulation through the cytoplasmic level of Pi.

Adenosine Triphosphate↗