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PubMed1991

DegS-DegU and ComP-ComA modulator-effector pairs control expression of the Bacillus subtilis pleiotropic regulatory gene degQ.

Production of a class of both secreted and intracellular degradative enzymes in Bacillus subtilis is regulated at the transcriptional level by a signal transduction pathway which includes the DegS-DegU two-component system and at least two additional regulatory genes, degQ and degR, encoding polypeptides of 46 and 60 amino acids, respectively. Expression of degQ was shown to be controlled by DegS-DegU. This expression is decreased in the presence of glucose and increased under any of the following conditions: growth with poor carbon sources, amino acid deprivation, phosphate starvation, and growth in the presence of decoyinine, a specific inhibitor of GMP synthetase. In addition, expression of degQ is shown to be positively regulated by the ComP-ComA two-component system. Separate targets for regulation of degQ gene expression by DegS-DegU and ComP-ComA were located by deletion analysis between positions -393 and -186 and between positions -78 and -40, respectively. Regulation of degQ expression by amino acid deprivation was shown to be dependent upon ComA. Regulation by phosphate starvation, catabolite repression, and decoyinine was independent of the two-component systems and shown to involve sequences downstream from position -78. The ComP-ComA and DegS-DegU two-component systems seem to be closely related, sharing several target genes in common, such as late competence genes, as well as the degQ regulatory gene. Sequence analysis of the degQ region revealed the beginning of an open reading frame directly downstream from degQ. Disruption of this gene, designated comQ, suggests that it also controls expression of degQ and is required for development of genetic competence.

Adenosine↗
PubMed1994

Cloning and sequencing the degS-degU operon from an alkalophilic Bacillus brevis.

The sacU region from an alkalophilic Bacillus brevis was cloned and sequenced. The two open reading frames of the degS-degU operon encode polypeptides that gave calculated molecular masses of 43.8 kDa and 27.0 kDa, respectively. Sequence comparisons at the amino acid level to the B. subtilis degS-degU genes showed 74% and 84% similarity, respectively. On a multicopy vector the B. brevis degS-degU genes were found to cause hypersecretion of several extracellular enzymes in a B. subtilis rec- strain as well as in a B. subtilis sacU(HY) strain.

Amino Acid Sequence↗
PubMed2002

Bacillus subtilis functional genomics: genome-wide analysis of the DegS-DegU regulon by transcriptomics and proteomics.

The DegS-DegU two-component regulatory system of Bacillus subtilis controls various processes that characterize the transition from the exponential to the stationary growth phase, including the induction of extracellular degradative enzymes, expression of late competence genes and down-regulation of the sigma(D) regulon. The degU32(Hy) mutation stabilizes the phosphorylated form of DegU (DegU-P), resulting in overproduction of several extracellular degradative enzymes. In this study, the pleiotropic DegS-DegU regulon was characterized by combining proteomic and transcriptomic approaches. A comparative analysis of wild-type B. subtilis and the degU32(Hy) mutant grown in complex medium was performed during the exponential and in the stationary growth phase. Besides genes already known to be under the control of DegU-P, novel putative members of this regulon were identified. Although the degU32(Hy) mutant is assumed to contain high levels of phosphorylated DegU in the exponential as well as in the stationary growth phase, many genes known to be positively regulated by DegU-P did not show enhanced expression in the mutant strain during exponential growth. This is consistent with the fact that most genes belonging to the DegS-DegU regulon are subject to multiple regulation; this is also reflected in the strong stationary-phase induction of these genes in the mutant strain. As expected, during the exponential growth phase, the sigma(D) regulon was expressed at significantly lower levels in the degU32(Hy) mutant than in the wild type.

Bacillus subtilis↗
PubMed2006

The role of the FtsH and Deg proteases in the repair of UV-B radiation-damaged Photosystem II in the cyanobacterium Synechocystis PCC 6803.

The photosystem two (PSII) complex found in oxygenic photosynthetic organisms is susceptible to damage by UV-B irradiation and undergoes repair in vivo to maintain activity. Until now there has been little information on the identity of the enzymes involved in repair. In the present study we have investigated the involvement of the FtsH and Deg protease families in the degradation of UV-B-damaged PSII reaction center subunits, D1 and D2, in the cyanobacterium Synechocystis 6803. PSII activity in a DeltaFtsH (slr0228) strain, with an inactivated slr0228 gene, showed increased sensitivity to UV-B radiation and impaired recovery of activity in visible light after UV-B exposure. In contrast, in DeltaDeg-G cells, in which all the three deg genes were inactivated, the damage and recovery kinetics were the same as in the WT. Immunoblotting showed that the loss of both the D1 and D2 proteins was retarded in DeltaFtsH (slr0228) during UV-B exposure, and the extent of their restoration during the recovery period was decreased relative to the WT. However, in the DeltaDeg-G cells the damage and recovery kinetics of D1 and D2 were the same as in the WT. These data demonstrate a key role of FtsH (slr0228), but not the Deg proteases, for the repair of PS II during and following UV-B radiation at the step of degrading both of the UV-B damaged D1 and D2 reaction center subunits.

Heat-Shock Proteins↗
PubMed1991

Thumb's rule tested: visual angle of thumb's width is about 2 deg.

Measures of the size of various hand parts and their viewing distance when held at arm's length were made on one hundred and eighteen undergraduate students. A simple rule of thumb can be confirmed: Visual angle of the width of the thumb held at arm's length is about 2 deg. The thumbnail subtends about 1.5 deg and the index fingernail about 1 deg in width, when both are held at arm's length. These figures are good approximations for males and females, although a significant, direct, linear relationship exists between hand-part size and the visual angle of the part at arm's length.

Adult↗
PubMed2004

Expression, crystallization and crystallographic analysis of DegS, a stress sensor of the bacterial periplasm.

Regulated proteolysis is a key event in transmembrane signalling between intercellular compartments. In Escherichia coli, a protein DegS has been identified as being a periplasmic stress sensor for unfolded or misfolded outer membrane proteins (OMPs). Activation of DegS initiates a proteolytic cascade which results in the transcription of periplasmic genes under sigmaE control, most importantly chaperones and proteases. DegS has been cloned and expressed as full-length protein and in an N-terminally truncated form. Both proteins were tested for crystallization and two forms of well diffracting crystals of the truncated form were obtained. Crystals of form I diffract to 3.5 A and belong to space group P2(1)3, while crystals of form II diffract to 2.2 A and belong to space group I23. Crystals of form II were soaked with a consensus peptide representing the C-termini of outer membrane proteins and data to 2.4 A resolution were collected. Molecular-replacement trials using a homologous protease domain indicate the presence of two molecules in the asymmetric unit of crystal form I. The correctness of the molecular-replacement solution was verified by identifying radiation-damage-induced structural changes.

Crystallization↗
PubMed1978

Deg phenotype of Escherichia coli lon mutants.

Deg. one of the Escherichia coli systems for degrading abnormal polypeptides (e.g., nonsense fragments), is also involved in the degradation of some classes of missense proteins. Both missense proteins of beta-galactosidase and temperature-sensitive phage products appear to be degraded by the Deg system. Mutations in the Deg system are indistinguishable from mutations classically called lon or capR; all map near proC, all are mucoid, defective in protein degradation, sensitive to radiomimetic agents, and defective in P1 lysogenization. All are able to propagate temperature-sensitive phage better than lon+ parental strains. Mutations that suppress the radiation sensitivity of these strains (sul) also suppress the P1 lysogenization defect, but do not affect mucoidy or the degradation defect.

Bacterial Proteins↗
PubMed1994

Multiple copies of the proB gene enhance degS-dependent extracellular protease production in Bacillus subtilis.

Bacillus subtilis secretes extracellular proteases whose production is positively regulated by a two-component regulatory system, DegS-DegU, and other regulatory factors including DegR. To identify an additional regulatory gene(s) for exoprotease production, we performed a shotgun cloning in the cell carrying multiple copies of degR and found a transformant producing large amounts of the exoproteases. The plasmid in this transformant, pLC1, showed a synergistic effect with multiple copies of degR on the production of the extracellular proteases, and it required degS for its enhancing effect. The DNA region responsible for the enhancement contained the proB gene, as shown by restriction analyses and sequence determination. The proB gene encoding gamma-glutamyl kinase was followed by the proA gene encoding glutamyl-gamma-semialdehyde dehydrogenase at an interval of 39 nucleotides, suggesting that the genes constitute an operon. pLC1 contained the complete proB gene and a part of proA lacking the proA C-terminal region. It was also found that proB on the chromosome showed a synergistic effect with multiple copies of degR. We consider on the basis of these results that the metabolic intermediate, gamma-glutamyl phosphate, would transmit a signal to DegS, resulting in a higher level of phosphorylated DegU. Possible involvement of DegR in this process is discussed.

1-Pyrroline-5-Carboxylate Dehydrogenase↗
PubMed1986

A 16 deg. wedge pad for urography.

With the patient supine the long axis of the kidney has an average 16 deg. (SD 5,8 deg.) distal ventral inclination to the horizontal table top plane. A 100 cm long wedge shaped pad with a 16 deg. angle to be placed under the patient's shoulders and back during urography to bring the long axis of the kidney parallel to the horizontal plane is described. This method gives simultaneous good visualization of both the upper and lower outlines of the kidneys on a single tomographic cut.

Humans↗
PubMed1991

Nephrotoxic effects of diethylene glycol (DEG) in rats.

Diethylene glycol (DEG) is a widely used substance with various risks of intoxication. In adult rats influences of DEG on functional parameters are characterized, indicating early signs of nephrotoxicity. A dose dependent proteinuria, an oliguric effect, an increased excretion of free hydrogen ions and a compensated impairment of renal tubular transport processes can be stated (0.25, 0.5 and 0.75 ml DEG/100 g b.m. i.p.). Following a single dose of 0.5 ml DEG/100 g b.m. i.p. the maximally expressed nephrotoxic effect is measurable 4 to 8 days after administration.

Animals↗
PubMed2000

Strengthening the dimerisation interface of Lac repressor increases its thermostability by 40 deg. C.

We increased drastically the heat stability of Lac repressor (LacR) of Escherichia coli. Wild-type tetrameric LacR denatures irreversibly at 53 degrees C. Improving hydrophobic packing at the dimerisation interface by a single substitution increases LacR heat-resistance by 40 deg. C without abolishing inducer binding at high and low temperatures. Tetrameric LacR mutants carrying substitutions of the positively charged amino acid Lys84 by each of the hydrophobic amino acids Leu, Ile and Met resist heating to temperatures up to 93 degrees C. We performed IPTG binding assays at 80 degrees C and found the mutant Lac repressors active and, thus, the core intact. Furthermore, the activity of LacR following heating is shown at room temperature by a gel retardation assay, which demonstrates normal oligomerisation state and function of the headpiece. The same mutations (K84L/I/M) in the dimer LacR331stop, carrying a stop codon in amino acid 331, increase thermostability of the dimer from 47 degrees C to 87 degrees C. LacRK84M represses beta-galactosidase activity in vivo as well as the wild-type and is sufficiently induced to allow growth on lactose. The results with both tetramer and dimer variants of LacR indicate mutual stabilisation of the tetramerisation region and the stable core.

Allosteric Regulation↗
PubMed1991

Diethylene glycol (DEG)-associated myocardial changes: a pilot investigation of chronic intoxication in guinea-pigs.

The myocardium of guinea pigs fed on sublethal doses of diethylene glycol (DEG) over a period of 2-11 days was examined for microscopical and ultrastructural changes. Coagulative myocytolysis and loss of myofibrils, not observed in the controls, was patchily distributed throughout the myocardium. The accompanying ultrastructural features included swelling, pleomorphism and hyperplasia of mitochondria with an associated distension of the interfibrillary spaces and a displacement, distortion and rupture of adjacent myofibrils.

Administration, Oral↗
PubMed2006

Application of an enzyme-linked immunosorbent assay for the analysis of imidacloprid in wiliwili tree, Erythrina sandwicensis O. Deg, for control of the wasp Quadrastichus erythrinae.

A monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) for the neonicotinoid insecticide imidacloprid was evaluated for its reproducibility, accuracy, and comparability to results from a conventional high-performance liquid chromatography (HPLC) for the analysis of imidacloprid in the endemic wiliwili tree (Erythrina sandwicensis O. Deg) found in dryland forests and landscapes in Hawaii. Imidacloprid was applied to these wiliwili trees in an attempt to control the newly introduced erythrina gall wasp, Quadrastichus erythrinae Kim. Leaf samples were freeze-dried and extracted with acidic aqueous methanol followed by methylene chloride partitioning. After solvent removal, the extract residue was reconstituted in 1 mL of water/methanol (1:1, v/v) for ELISA; no significant matrix interference was observed at 10-fold or more dilution. The average recoveries of imidacloprid from fortified samples ranged from 78% to 100% by ELISA. The correlation between the ELISA and HPLC results was excellent (r2 = 0.98). Imidacloprid was detected with the ELISA in all treated samples and its level varied in the samples among different treatments and in those from different parts of the trees. The infestation severity rating of leaf samples was inversely related to the concentration of imidacloprid. It is clear that imidacloprid effectively controls the wasps. The ELISA is a suitable method for quantitative and reliable determination of imidacloprid in wiliwili trees and the application provides information to understand how to control the wasps.

Chromatography, High Pressure Liquid↗
PubMed1986

Interindividual receptor variability of normal colour observers: analysis of the 2-deg Stiles and Burch data.

Examination of the interobserver variability among the ten observers of the 2-deg Stiles and Burch colorimetric study reveals an interesting pattern: the chromaticities of the matches made by different normal subjects fall approximately on straight lines and these lines appear to converge. Because of a geometric relationship that holds between the normal, anomalous, and dichromatic colour spaces, it can be argued that part of the variability must be due to actual differences in the shape or spectral position of the receptor sensitivities of different normal observers.

Adult↗
PubMed1989

Role of adenovirus type 2 early region 1B 19K protein stability in expression of the cyt and deg phenotypes.

The cell line KB18 constitutively expresses adenovirus type 2 (Ad2) early region 1B (E1B) genes in the absence of E1A expression and thus is useful for understanding the function and properties of E1B gene products. We report here that KB18 cells complement the cyt and deg phenotypes of Ad2 cytocidal (cyt) mutants and an Ad12 cyt mutant. Thus the E1B 19K polypeptide in KB18 is functional. Expression of E1B mRNA and synthesis of the 19K polypeptide were studied in wild-type Ad2- and Ad2cyt15-infected KB cells at 15 h post-infection, and in KB18 cells. Although E1B mRNA synthesized in KB18 cells at 5 to 7% of the level in Ad2-infected KB cells, the amount of E1B 19K polypeptide formed was similar. In contrast, the amount of 19K protein in Ad2cyt15-infected KB cells was about one-tenth of that in Ad2-infected cells, although expression of E1B mRNA was comparable to that during Ad2 infection. The half-lives of the 19K polypeptide in wild-type-infected KB cells, in Ad2cyt15-infected KB cells and in KB18 were approximately 90 min, 25 min and 22 h, respectively. The cyt phenotype was expressed at 30 to 35 h post-infection, thus showing that the instability of the 19K polypeptide in Ad2cyt15-infected cells is not due to alteration of cell morphology or cell destruction.

Adenovirus Early Proteins↗
PubMed2002

DegS and YaeL participate sequentially in the cleavage of RseA to activate the sigma(E)-dependent extracytoplasmic stress response.

All cells have stress response pathways that maintain homeostasis in each cellular compartment. In the Gram-negative bacterium Escherichia coli, the sigma(E) pathway responds to protein misfolding in the envelope. The stress signal is transduced across the inner membrane to the cytoplasm via the inner membrane protein RseA, the anti-sigma factor that inhibits the transcriptional activity of sigma(E). Stress-induced activation of the pathway requires the regulated proteolysis of RseA. In this report we show that RseA is degraded by sequential proteolytic events controlled by the inner membrane-anchored protease DegS and the membrane-embedded metalloprotease YaeL, an ortholog of mammalian Site-2 protease (S2P). This is consistent with the mechanism of activation of ATF6, the mammalian unfolded protein response transcription factor by Site-1 protease and S2P. Thus, mammalian and bacterial cells employ a conserved proteolytic mechanism to activate membrane-associated transcription factors that initiate intercompartmental cellular stress responses.

Activating Transcription Factor 6↗
PubMed1993

Assembly of in Vitro-Synthesized Large Subunits into Ribulose Bisphosphate Carboxylase/Oxygenase Is Sensitive to CI-, Requires ATP, and Does Not Proceed When Large Subunits Are Synthesized at Temperatures [greater than or equal to]32[deg]C.

In higher plants, ribulose bisphosphate carboxylase/oxygenase (Rubisco) consists of eight large "L" subunits, synthesized in chloroplasts, and eight small "S" subunits, synthesized as precursors in the cytosol. Assembly of these into holoenzyme occurs in the chloroplast stroma after import and processing of the S subunits. A chloroplast chaperonin interacts with the L subunits, which dissociate from the chaperonin before they assemble into holoenzyme. Our laboratory has reported L subunit assembly into Rubisco in chloroplast extracts after protein synthesis in leaves, intact chloroplasts, and most recently in membrane-free chloroplast extracts. We report here that the incorporation of in vitro-synthesized L subunits into holoenzyme depends on the conditions of L subunit synthesis. Rubisco assembly did not occur after L subunit synthesis at 160 mM KCI. When L subunit synthesis occurred at approximately 70 mM KCI, assembly depended on the temperature at which L subunit synthesis took place. These phenomena were the result of postsynthetic events taking place during incubation for protein synthesis. We separated these events from protein synthesis by lowering the temperature during protein synthesis. Lower temperatures supported the synthesis of full-length Rubisco L subunits. The assembly of these completed L subunits into Rubisco required intervening incubation with ATP, before addition of S subunits. ATP treatment mobilized L subunits from a complex with the chloroplast chaperonin 60 oligomer. Addition of 130 mM KCI at the beginning of the intervening incubation with ATP blocked the incorporation of L subunits into Rubisco. The inhibitory effect of high KCI was due to CI- and came after association of newly synthesized L subunits with chaperonin 60, but before S subunit addition. It is interesting that L subunits synthesized at [greater than or equal to]32[deg]C failed to assemble into Rubisco under any conditions. These results agree with previous results obtained in this laboratory using newly synthesized L subunits made in intact chloroplasts. They also show that assembly of in vitro-synthesized L subunits into Rubisco requires ATP, that CI- inhibits Rubisco assembly, and that synthesis temperature affects subsequent assembly competence of L subunits.

Journal Article↗
PubMed1994

Sinusoidal variation of wall shear stress in daughter tube through 45 deg branch model in laminar flow.

The present paper describes the experimental implemented work on the flow situation through a branch model having a daughter tube bifurcated from a parent tube at 45 deg. Experiments have been conducted utilizing an electrochemical method. The results show that, even in steady flow, the wall shear stress along the proximal wall in the daughter tube varies significantly with position in the form of a damped sine wave. For pulsating flow at the nondimensional pulsating frequency of alpha = 6.6, the above mentioned tendency appears to be severe, and the distribution of its amplitude in pulsating flow is similar to that of wall shear stress in steady flow.

Aorta, Abdominal↗