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PubMed2000

Trichloroethylene degradation by Ralstonia sp. KN1-10A constitutively expressing phenol hydroxylase: transformation products, NADH limitation, and product toxicity.

Ralstonia sp. KN1-10A, which was constructed by inserting the tac promoter upstream of the phenol hydroxylase (PH) gene in the chromosomal DNA of the wild-type strain, Ralstonia sp. KN1, is a useful recombinant strain for eliminating trichloroethylene (TCE) from contaminated sites because it exhibits constitutive TCE oxidation activity. During TCE degradation by Ralstonia sp. KN1-10A, noxious chlorinated compounds, such as dichloroacetic acid, trichloroacetic acid, 2,2,2-trichloroethanol, and chloral, were not detected, and more than 95% of chlorine in TCE was released as chloride ions. Among the possible TCE transformation products, only carbon monoxide was detected, and its conversion percentage was 7 mol%. The addition of formate, which Ralstonia sp. KN1-10A could use as an exogenous electron donor, did not enhance the TCE degradation performance, suggesting that NADH depletion did not limit the degradation. The phenol degradation activity of Ralstonia sp. KN1-10A that previously degraded TCE was not markedly lower than that of cells not exposed to TCE, suggesting that Ralstonia sp. KN1-10A was not susceptible to product toxicity associated with TCE degradation. Furthermore, to clarify the mechanisms underlying TCE degradation by PH from Ralstonia sp. KN1, this enzyme was compared with another enzyme, a hybrid aromatic ring dioxygenase exhibiting a high TCE degradation activity in Escherichia coli and Pseudomonas sp. The initial TCE degradation rate of Ralstonia sp. KN1 (pKTP100), which produced PH, was 1 50 lower than that of Ralstonia sp. KN1 (pKTF200), which produced the hybrid aromatic ring dioxygenase. However, because of its lower product toxicity, the strain producing PH could degrade 2.3 times more TCE than that generated by the strain producing the hybrid aromatic ring dioxygenase.

Journal Article↗
PubMed2004

Stage-specific and species-specific differences in the production of the mRNA and protein for the filarial nematode secreted product, ES-62.

Previous studies have shown that the secreted phosphorylcholine-containing glycoprotein of filarial nematodes, ES-62, is only present in the post-infective life-cycle stages, but that the mRNA is transcribed throughout the worm's life-cycle. The aim of this current study was to investigate whether the presence or absence of protein expression simply reflects differences in mRNA abundance. To this end, we investigated the relative abundance of ES-62 using TaqMan real time RT-PCR, in different life-cycle stages of 2 model filarial nematode parasites, Acanthocheilonema viteae and Brugia pahangi. For B. pahangi, microfilariae, infective larvae and adult worms were each found to have approximately similar levels of ES-62 mRNA. However, the corresponding stages of A. viteae differed greatly from each other with a pattern of increased mRNA production with maturation. As a rule A. viteae had higher levels of ES-62 mRNA than B. pahangi, and this was particularly noticeable in the adult stage where the difference was approximately 3500-fold higher. However, this significant difference in mRNA abundance was not reflected in the quantity of ES-62 protein secreted by the adult worms of each species, as A. viteae only secreted approximately 3 times as much ES-62 as B. pahangi. Thus, overall, the results obtained from this study indicate that ES-62 protein production does not solely reflect mRNA levels, and also suggest that the 2 nematodes may employ different mechanisms for regulating protein production.

Animals↗
PubMed2002

High-throughput method for the production and analysis of large natural product libraries for drug discovery.

High-throughput methods were applied to the production, analysis, and characterization of libraries of natural products in order to accelerate the drug discovery process for high-throughput screening in the pharmaceutical and biotechnology industries. Library production integrates automated flash chromatography, solid-phase extraction, filtration, and high-throughput parallel four-channel preparative high-performance liquid chromatography to obtain the libraries in 96- or 384-well plates. Libraries consist of purified fractions with approximately one to five compounds per well. Libraries are analyzed prior to biological screening by a high-throughput parallel eight-channel liquid chromatography-evaporative light scattering detection-mass spectrometry system to determine the molecular weight, number, and quantity of compounds in a fraction. After biological screening, active fractions are rapidly purified at the microgram level and individual compounds are rescreened for confirmation of activity. Structures of active compounds are elucidated by NMR spectroscopy and mass spectrometry. Utilization of a novel microcoil probe allows NMR data to be gathered on 50 microg. As a demonstration, a library was made from the stem bark of Taxus brevifolia. Biological screening in the National Cancer Institute's in vitro panel of three cancer cell lines demonstrates that the process enables the discovery of active anticancer compounds not detected in the flash fractions from which the library originates.

Biological Factors↗
PubMed1988

Inhibition of cell-mediated immunity by tumour cell products: depression of interleukin-2 production and responses to interleukin-2 by mouse spleen cells.

Supernatants from cultures of mouse and human tumour cells inhibited the production of interleukin-2 (IL-2) by stimulated mouse spleen cells. The tumour cells tested, all of which were active, included a mouse and a human melanoma, three methylcholanthrene-induced fibrosarcomas of mice, and human HeLa cells. Supernatants from normal mouse and human fibroblasts were inactive. Inhibition was dose-dependent. Spleen cells from aged mice were more susceptible to inhibition than spleen cells from young mice. When tumour cell culture supernatants were fractionated on Sephacryl S-300, two peaks of activity were found, with apparent molecular weights of approximately 50 and 18 kD. Supernatants from tumour cell and fibroblast cultures caused variable, but generally weak, inhibition of responses of lymphoblasts to IL-2. It is suggested that inhibition of IL-2 production may be an important mode of action of tumour cell products that inhibit cell-mediated immunity.

Animals↗
PubMed2006

Chitinase inhibitor allosamidin is a signal molecule for chitinase production in its producing Streptomyces I. Analysis of the chitinase whose production is promoted by allosamidin and growth accelerating activity of allosamidin.

Allosamidin, a typical secondary metabolite of Streptomyces, has been known as a chitinase inhibitor. We found that allosamidin can dramatically promote chitinase production and growth of its producer, Streptomyces sp. AJ9463, in a chitin medium at a few hundred nM. Allosamidin promoted production of the main chitinase detected in the culture filtrate and the chitin-hydrolytic activity of the chitinase was not inhibited by allosamidin at the concentration. The gene encoding the chitinase showed that it is a family 18 chitinase and it was revealed that two genes encoding proteins constructing two-component regulatory system were present at 5'-upstream region of the chitinase gene. Allosamidin is located in the microbial mycelia cultured in a medium without chitin, but it was released from the mycelia by responding to chitin. These results show that allosamidin acts as a key signal molecule for chitinase production in its producing strain, which may be useful for its growth in chitin-rich environment.

Acetylglucosamine↗
PubMed1993

Formic acid is a product of the alpha-oxidation of fatty acids by human skin fibroblasts: deficiency of formic acid production in peroxisome-deficient fibroblasts.

Human skin fibroblasts in culture can oxidize beta-methyl fatty acids, such as phytanic acid and 3-methylhexadecanoic acid, to CO2 and water-soluble products. The latter are released largely into the culture medium. The major water-soluble product formed from [1-14C]phytanic and [1-14C]3-methylhexadecanoic acids is [14C]formic acid. As phytanic acid and 3-methylhexadecanoic acids contain beta-methyl groups and theoretically cannot be degraded by beta-oxidation, we postulate that formic acid is formed from fatty acids by alpha-oxidation. The marked reduction in formic acid production from beta-methyl fatty acids in peroxisome-deficient skin fibroblasts suggests that peroxisomes are involved in the generation of C1 units.

Cells, Cultured↗
PubMed1994

Kinetics of an enzyme reaction in which both the enzyme-substrate complex and the product are unstable or only the product is unstable.

A kinetic analysis of the Michaelis-Menten mechanism has been made for the case in which both the enzyme-substrate complex and the product are unstable or only the product is unstable, either spontaneously or as the result of the addition of a reagent. This analysis allows the derivation of equations which under conditions of limiting enzyme concentration relate the concentration of all of the species to the time. A kinetic data analysis is suggested, which leads to the evaluation of the kinetic parameters involved in the reaction. The analysis is based on the equation which describes the formation of products with time and one's experimental progress curves. We demonstrate the method numerically by computer simulation of the reaction with added experimental errors and experimentally by the use of data from the kinetic study of the action of tyrosinase on dopamine.

Basidiomycota↗
PubMed2001

Lactate dehydrogenase has no control on lactate production but has a strong negative control on formate production in Lactococcus lactis.

A series of mutant strains of Lactococcus lactis were constructed with lactate dehydrogenase (LDH) activities ranging from below 1% to 133% of the wild-type activity level. The mutants with 59% to 133% of lactate dehydrogenase activity had growth rates similar to the wild-type and showed a homolactic pattern of fermentation. Only after lactate dehydrogenase activity was reduced ninefold compared to the wild-type was the growth rate significantly affected, and the ldh mutants started to produce mixed-acid products (formate, acetate, and ethanol in addition to lactate). Flux control coefficients were determined and it was found that lactate dehydrogenase exerted virtually no control on the glycolytic flux at the wild-type enzyme level and also not on the flux catalyzed by the enzyme itself, i.e. on the lactate production. As expected, the flux towards the mixed-acid products was strongly enhanced in the strain deleted for lactate dehydrogenase. What is more surprising is that the enzyme had a strong negative control ( CLDHJF1 =-1.3) on the flux to formate at the wild-type level of lactate dehydrogenase. Furthermore, we showed that L. lactis has limited excess of capacity of lactate dehydrogenase, only 70% more than needed to catalyze the lactate flux in the wild-type cells.

Fermentation↗
PubMed2002

Spontaneous production of RANTES and antigen-specific IFN-gamma production in macaques vaccinated with SHIV-4 correlates with protection against SIVsm challenge.

The beta-chemokines, RANTES, MIP-1alpha and MIP-1beta, have been implicated as being some of the protective factors in the immune response against human immunodeficiency virus (HIV) infection. We have presented data previously indicating that these chemokines also play a role in protective immunity against HIV/SIV infection in macaques. The aim of this study was to investigate the production of beta-chemokines in eight cynomolgus macaques vaccinated with non-pathogenic SHIV-4 in relation to protection against pathogenic SIVsm challenge. Four control animals were also included in the study. Two of the vaccinated monkeys were completely protected and one was partially protected against the challenge virus. The monkeys that resisted infectious SIVsm virus challenge showed higher spontaneous beta-chemokine production by peripheral blood mononuclear cells and had higher numbers of antigen-induced IFN-gamma secreting cells compared to the non-protected animals. Our observations support our previous findings that the genetic background of the host and/or environmental factors are involved in the chemokine production and that beta-chemokines contribute to protection against HIV/SIV infection.

Animals↗
PubMed1998

Increased circulating cytokine receptors and ex vivo interleukin-1 receptor antagonist and interleukin-1beta production but decreased tumour necrosis factor-alpha production after a 5-km run.

BACKGROUND: The purpose of this study was to examine the effect of a 5-km run on blood leucocytes, acute-phase proteins and cytokines. In addition, cytokines were measured in the supernatants from whole-blood cell cultures incubated with lipolysaccharide (LPS). METHODS: Ten healthy, recreational trained, athletes (three women, seven men) volunteered for this investigation. Samples were drawn just before, immediately after and at 3 h, at 24 h and at 48 h after the race. RESULTS: Exercise induced a transient leucocytosis (P = 0. 0002) and a mild acute-phase reaction with increase in plasma C-reactive protein (CRP) (P = 0.0115) but not in serum amyloid A (SAA) concentrations. Although plasma interleukin 6 (IL-6) was undetectable and soluble interleukin-1 receptor type II (IL-1sRII) remained unchanged, interleukin-1 receptor antagonist (IL-1ra) concentrations were elevated directly after the race with a further increase at 3 h (P < 0.0001). Soluble tumour necrosis factor (TNF) receptors were increased immediately after the run, but the effect was more marked for sTNFr p55 (two-fold increase; P < 0.0001) than for sTNFr p75 (1.16-fold increase; P = 00007). In cell cultures, the LPS-induced release of the inflammatory cytokines doubled for IL-1beta (P < 0.0001) and for IL-1ra (P < 0.0001). In contrast, TNF-alpha production decreased after the run, and a nadir was reached at 24 h (P < 0.0001). CONCLUSION: These results suggest that a 5-km run elicits both the production of acute-phase mediators (leucocytosis and elevation of CRP) and anti-inflammatory counter-regulation as judged by the increase in circulating concentrations of IL-1ra, sTNFr p55, and sTNFrp75 and down-regulation of LPS-stimulated TNF-alpha production.

Adolescent↗
PubMed1999

Production of lysozyme-enriched biomass from cheese industry by-products.

Cheese whey and cottage cheese whey are by-products of the milk and cheese industry, resulting from the production of cheese and cottage cheese (ricotta) from milk. They are still rich in organic substances and cannot be discarded into the environment without proper treatment. Whey and cottage cheese whey were used as culture media for some strains of the yeast Kluyveromyces lactis, transformed with the human lysozyme gene. It was found that the yeast strains grew well in both media and produced a considerable amount of recombinant protein. Production kinetics showed that the human lysozyme was produced in a greater amount within 36 h of fermentation (125 micrograms ml-1 vs 25 micrograms ml-1 in the control) than in the synthetic commercial media used for strain preparation and characterization. The recombinant protein produced was actually shown to be the human lysozyme, using renaturing SDS-PAGE and Western blot techniques. While producing recombinant protein, the Kluyveromyces strain cleared the cottage cheese whey of most organic substances and produced a considerable amount (almost 3%) of lysozyme-enriched useful biomass.

Animals↗
PubMed2000

Carcass quality in certified organic production compared with conventional livestock production.

By studying carcass quality, expressed as affection, pathological findings, slaughter-weight and evaluation, a picture of an animal's health and potential as high quality food is achieved. This study compares the carcass quality in Swedish certified organic meat production with that of conventional meat production slaughtered during 1997. The study involves 3.9 million pigs, about 570,000 cattle and 190,000 sheep, all reared conventionally and 3483 pigs 4949 cattle and 4997 sheep reared according to organic standards. Pathological and additional findings are registered by meat inspectors from the Swedish National Food Administration at the post-mortem inspection. There was a significant difference at the post-mortem inspection of growing-fattening pigs; 28% of conventionally and 17% of the organically reared pigs had one or more registered lesion. The carcass evaluation of swine shows a higher meat percentage in conventional swine production. The total rate of registered abnormalities in cattle was systems around 28% from organic and 27% from conventionally reared herds. Carcass evaluation of cattle from organic herds gave higher classification in the EUROP system, whereas the fat content was lower than that of conventionally reared cattle. Sheep, reared both organically and conventionally, showed a lower rate of registered abnormalities than swine and cattle.

Animals↗
PubMed1984

Pulmonary excretion of carbon monoxide in the human infant as an index of bilirubin production. IIc. Evidence for the possible association of cord blood erythropoietin levels and postnatal bilirubin production in infants of mothers with abnormalities of gestational glucose metabolism.

A total of 20 infants who had levels of erythropoietin (Ep), the major hormone regulating erythropoiesis, measured in their cord blood also had determinations of the pulmonary excretion rate of CO (VECO) performed, as an index of total bilirubin production. They were either infants of normal mothers or those of mothers with diabetes, gestational diabetes, and missed abnormalities of gestational glucose metabolism. The mean VECO (13.0 +/- 3.5 mu 1/kg/hr) and the mean Ep (20.0 +/- 9.7 SD mU/ml) of the infants with normal mothers (n = 9) were not different from the means previously established by our laboratories (13.9 +/- 3.5 SD mu 1/kg/hr, n = 20; and 23.7 +/- 12.8 SD mU/ml, n = 30, respectively); they were significantly lower than those of the infants of the abnormal mothers in this study. The 5 infants who had a cord blood Ep level greater than 50 mU/ml had a higher mean VECO, 27.8 +/- 7.1 mu 1/kg/hr, compared with 17.2 +/- 4.9 SD mu 1/kg/hr, of the six infants with cord blood Ep levels that were within 2 SD of the previously established normal mean cord blood Ep level (p less than .025). These data suggest that increased cord blood Ep levels and postnatal bilirubin production in infants whose mothers had abnormalities of gestational glucose metabolism are associated phenomena. Since polycythemia did not occur in these infants, ineffective erythropoiesis or mild, compensated hemolysis remains a likely cause of the increased total bilirubin production. In some cases, perinatal hypoxic stress may have affected the Ep response.(ABSTRACT TRUNCATED AT 250 WORDS)

Bilirubin↗
PubMed1984

Attenuation of the ilvB operon by amino acids reflecting substrates or products of the ilvB gene product.

Transcription termination at the ilvB attenuator of Escherichia coli K-12 has been quantitated by measuring the in vivo rate of synthesis and degradation of mRNA segments proximal and distal to the attenuator. This analysis demonstrates that a 4-fold deattenuation results in vivo when the growth of cells is limited by the availability of valine or leucine. The result suggests that attenuation is the major mechanism by which this operon is regulated by these end-product amino acids. On the basis of possible secondary structures of ilvB leader RNA, we predicted that attenuation of this operon should also be affected by growth of cells in limiting amounts of alanine or threonine. We report here that the ilvB operon is deattenuated when cells are starved for either of these amino acids. A rationale for the regulation of this operon by these four amino acids, which represent both the substrates and the products of the ilvB gene product, and by catabolite repression is presented.

Bacterial Proteins↗
PubMed1985

Products of nitrogen regulatory genes ntrA and ntrC of enteric bacteria activate glnA transcription in vitro: evidence that the ntrA product is a sigma factor.

In enteric bacteria the products of two nitrogen regulatory genes, ntrA and ntrC, activate transcription of glnA, the structural gene encoding glutamine synthetase, both in vivo and in vitro. The ntrC product (gpntrC) is a DNA-binding protein, which binds to five sites in the glnA promoter-regulatory region and appears to activate transcription initiation. Using as an assay the stimulation of glnA transcription in a coupled in vitro transcription-translation system, we have partially purified the ntrA gene product (gpntrA). The following evidence is consistent with the view that gpntrA is a sigma subunit for RNA polymerase: (i) The gpntrA activity copurifies with the sigma 70 holoenzyme (E sigma 70) and core (E) forms of RNA polymerase through several steps but can be separated from them by chromatography on heparin agarose. (ii) After further purification by molecular sieve chromatography, the partially purified gpntrA fraction allows transcription of glnA from the same startpoint used in vivo; transcription is dependent on gpntrC and on added E. The gpntrA fraction does not allow transcription from promoters that we have used as controls, including lacUV5. E sigma 70 has the reverse specificity.

Bacterial Proteins↗
PubMed2001

L-arginine chlorination products inhibit endothelial nitric oxide production.

The myeloperoxidase-derived oxidant hypochlorous acid (HOCl) is thought to contribute to endothelial dysfunction, but the mechanisms underlying this inhibitory effect are unknown. The present study tested the hypothesis that HOCl and L-arginine (L-Arg) react to form novel compounds that adversely affect endothelial function by inhibiting nitric oxide (NO) formation. Using spectrophotometric techniques, we found that HOCl and L-Arg react rapidly (k = 7.1 x 10(5) m(-1) s(-1)) to form two major products that were identified by mass spectrometry as monochlorinated and dichlorinated adducts of L-Arg. Pretreatment of bovine aortic endothelial cells with the chlorinated L-Arg metabolites (Cl-l-Arg) inhibited the -induced formation of the NO metabolites nitrate (NO(3)(-)) and nitrite (NO(2)(-)) in a concentration-dependent manner. Preincubation of rat aortic ring segments with Cl-L-Arg resulted in concentration-dependent inhibition of acetylcholine-induced relaxation. In contrast, blood vessels relaxed normally to the endothelium-independent vasodilator sodium nitroprusside. In vivo administration of Cl-L-Arg to anesthetized rats increased carotid artery vascular resistance. A greater than 10-fold excess of L-Arg was required to reverse the inhibitory effects of Cl-L-Arg in vivo and in vitro. Reaction of HOCl with D-arginine (D-Arg) did not result in the formation of inhibitory products. These results suggest that HOCl reacts with L-Arg to form chlorinated products that act as nitric-oxide synthase inhibitors.

Animals↗
PubMed2002

Operating the production calculus: ordering a production system in the print industry.

The topic of shop-floor work has been extensively examined within sociology. However, the organizational structures within which this work takes place have, in the most part, been taken as unexamined givens. Yet, their operation is also the shop-floor work of some people. This paper examines the way in which the stable organizational structures within which shop-floor work takes place are achieved. It is based upon a fieldwork investigation of a large commercial printer and focuses upon the collaborative work of those who are involved in scheduling the production of a job and their use of 'the production calculus' in planning the work of the site. The print industry is undergoing considerable technological change and scheduling technologies have been developed to automate this work. However, there has been little take up of these technologies and the paper also considers how the characteristics of operating the production calculus in practice may account for this.

Humans↗
PubMed1995

Effect of restricted feeding during the rearing period and a "forced moult" at 40 weeks of production on the productivity of Pekin breeder ducks.

1. Six males and 24 female Cherry Valley Pekin ducklings were randomly allocated to each of 12 pens to test the effects of quantitative food restriction during the rearing period on the subsequent reproductive performance. At the end of the laying period (60 weeks) a "forced moult" was introduced and its effect on a further 40 weeks production investigated. 2. The 3 rearing treatments were 50% of ad libitum intake from 3 to 20 weeks; 75% of ad libitum intake from 3 to 20 weeks and ad libitum intake throughout. During the laying period (20-60 weeks) and post forced-moult period (60-100 weeks) duck breeder pellets were fed ad libitum to all groups. 3. Highly significant (P < or = 0.01) differences in favour of the restricted groups over the ad libitum-fed groups were observed in most of the responses studied. The following were the main advantages of food restriction: lower lifetime food intake, lower carcase fat content at 20 weeks, delay in sexual maturity, higher egg production, higher peak and terminal production, higher fertility, higher hatchability of eggs set and lower mortality. Egg mass and hatchability of fertile eggs was not influenced by food restriction in this experiment. The use of a "forced moult" showed that it could be of economical importance. 4. Difference between treatments in respect of absolute and relative visceral masses were also investigated. The absolute masses of the intestine, heart, gizzard and liver all decreased as the degree of food restriction increased. The relative visceral masses showed the opposite trend with the exception of the heart which showed no significant differences between treatments.

Aging↗