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PubMed2004

Iron supplementation in adolescent hemodialysis patients.

AIMS: Iron supplementation is necessary in children on hemodialysis, but the optimal protocol remains unknown. We studied the effects of changing our unit's protocol from oral iron with periodic doses of parenteral iron dextran to routine administration of parenteral sodium ferric gluconate on anemia and iron parameters. METHODS: We followed seven hemodialysis patients aged 15 20 years (mean 17 years). Hemoglobin, hematocrit, serum iron, transferrin saturation, ferritin, erythropoietin dose, total elemental iron dose and total iron cost for the six months prior to the protocol change were compared to the same variables during the six months following the change. RESULTS: There was no statistically significant difference between the doses of parenteral iron between the two protocols; however, the total dose of elemental iron administered in the oral iron plus iron dextran protocol was greater than in the sodium ferric gluconate protocol (19.6+/-13.1 (mean+/-SD) mg/kg/week versus 1.1+/-0.3 mg/kg/week; p = 0.03). Both protocols had equivalent efficacy with respect to hemoglobin, ferritin and other measures of iron stores. On the other hand, the costs of sodium ferric gluconate were significantly higher than those of oral iron plus intermittent iron dextran (157.75+/-45.94 $/patient/month versus 52.08+/-49.88 $/patient/month; p = 0.01). CONCLUSIONS: Routine administration of sodium ferric gluconate is equivalent if not superior to use of oral iron plus iron dextran for maintenance of iron stores in adolescents on hemodialysis, but more expensive.

Administration, Oral↗
PubMed1990

In vivo metabolism of 3-deoxy-3-fluoro-D-glucose.

Recent studies have demonstrated that 3-deoxy-3-fluoro-D-glucose (3-FG) is metabolized to 3-deoxy-3-fluoro-D-sorbitol (3-FS), via aldose reductase, and 3-deoxy-3-fluoro-D-fructose (3-FF), via the sorbitol dehydrogenase reaction with 3-FS, in rat cerebral tissue (Kwee, I. L., Nakada, T., and Card, P. J. (1987) J. Neurochem. 49, 428-433). However, the biochemistry of 3-FG in other mammalian organs has not been investigated making the application of 3-FG as a metabolic tracer uncertain. To address this issue we investigated 3-FG metabolism and distribution in isolated cell lines and in rabbit tissues in vivo with 19F NMR and gas chromatography-mass spectrometry. In general, the production of 3-FS is well correlated with the known distribution of aldose reductase in all the systems studied. Further metabolism of 3-FS to 3-FF was verified to occur in cerebral tissue. Surprisingly, two new fluorinated compounds were found in the liver and kidney cortex. These compounds are identified as 3-deoxy-3-fluoro-D-gluconic acid, which is produced via glucose dehydrogenase activity on 3-FG, and 3-deoxy-3-fluoro-D-gluconate-6-phosphate. Based on enzyme studies, it is argued that the 3-deoxy-3-fluoro-D-gluconate-6-phosphate is derived directly from 3-deoxy-3-fluoro-D-gluconic acid and not as a product of pentose phosphate activity. Direct oxidation and reduction are the major metabolic routes of 3-FG, not metabolism through glycolysis or the pentose phosphate shunt. Thus, 3-FG metabolism coupled with 19F NMR appears to be very useful for monitoring aldose reductase and glucose dehydrogenase activity in vivo.

Aldehyde Reductase↗
PubMed1986

Stability of amiodarone hydrochloride in admixtures with other injectable drugs.

The stability of amiodarone hydrochloride in intravenous admixtures was studied. Amiodarone hydrochloride 900 mg was mixed with 500 mL of either 5% dextrose injection or 0.9% sodium chloride injection in polyvinyl chloride or polyolefin containers; identical solutions were also mixed with either potassium chloride 20 meq, lidocaine hydrochloride 2000 mg, quinidine gluconate 500 mg, procainamide hydrochloride 2000 mg, verapamil hydrochloride 25 mg, or furosemide 100 mg. All admixtures were prepared in triplicate and stored for 24 hours at 24 degrees C. Amiodarone concentrations were determined using a stability-indicating high-performance liquid chromatographic assay immediately after admixture and at intervals during storage. Each solution was visually inspected and tested for pH. Amiodarone concentrations decreased less than 10% in all admixtures except those containing quinidine gluconate in polyvinyl chloride containers. The only visual incompatibility observed was in admixtures containing quinidine gluconate and 5% dextrose injection. In most solutions pH either decreased slightly or remained unchanged. Amiodarone hydrochloride is stable when mixed with either 5% dextrose injection or 0.9% sodium chloride injection in polyvinyl chloride or polyolefin containers alone or with potassium chloride, lidocaine, procainamide, verapamil, or furosemide and stored for 24 hours at 24 degrees C. Amiodarone should not be mixed with quinidine gluconate in polyvinyl chloride containers.

Amiodarone↗
PubMed1978

Comparative evaluation of surgical scrub preparations.

The efficacy of 0.75 per cent available povidone-iodine scrub solution, 4 per cent chlorhexidine gluconate detergent solution and 3 per cent hexachlorophene emulsion against resident and transient flora of the hand has been compared using two currently accepted study designs. Chlorhexidine gluconate produced the greatest initial reductions against resident flora, followed by povidone-iodine and hexachlorophene, respectively. In subsequent washings, all three treatments further reduced the flora, chlorhexidine giving the greatest reduction on any test day. On gloved hands, there was significant regrowth following the use of povidone-iodine; there was no such significant regrowth on gloved hands which had been washed with chlorhexidine gluconate or hexachlorophene. Against transient flora, chlorhexidine gluconate gave the lowest over-all counts, followed by povidone-iodine and hexachlorophene, respectively. The reductions in bacteria counts increased following the repetitive use of chlorhexidine; no such trend was noted with either povidone-iodine or hexachlorophene.

Anti-Infective Agents, Local↗
PubMed2005

The effects of various organic acids on phytate phosphorus utilization in chicks.

Previous research from our laboratory has shown that citric acid improves phytate P utilization in chicks fed a P-deficient corn-soybean meal diet. The current study was conducted to determine if other organic acids also are effective, with an emphasis on gluconic acid. Four experiments were conducted in which 4 replicate groups of 5 crossbred chicks (New Hampshire x Columbian) were fed a P-deficient diet (0.16% nonphyate P) from 8 to 22 d of age. In Experiment 1, chick weight gain and tibia ash were significantly increased (P < 0.05) by 1.5 and 3% sodium gluconate (NaGlu), 1.5% calcium gluconate (CaGlu), 1.5 and 3% glucono-delta-lactone, and 1% 2-hydroxy-4-methylthio butanoic acid (Alimet). In experiment 2, tibia ash was significantly increased (P < 0.05) by 2% NaGlu, CaGlu, and citric acid in chicks fed the P-deficient diet but not in chicks fed a 0.45% nonphytate P diet, indicating that the organic acid responses were due to increased P utilization. In experiments 3 and 4, tibia ash was significantly increased by 3% NaGlu and 3% citric acid, but not by 3% fumaric acid or 0.025, 0.05 and 0.1% EDTA. The results of this study showed that NaGlu, CaGlu, glucono-delta-lactone, Alimet, and citric acid, but not fumaric acid or EDTA, improved phytate P utilization in chicks fed a corn-soybean meal diet.

Animals↗
PubMed1963

KINASES OF STREPTOCOCCUS FAECALIS. I. ENZYME LOCALIZATION.

Moore, L. Daneo (University of Pennsylvania, Philadelphia) and D. J. O'Kane. Kinases of Streptococcus faecalis. I. Enzyme localization. J. Bacteriol. 86:766-772. 1963.-Cell-free extracts of Streptococcus faecalis 10C1 were found to contain a membrane-associated, particulate hexokinase with activity toward glucose, fructose, mannose, and, to a lesser extent, galactose, gluconate, and mannitol. In addition, the cell cytoplasm contained kinases specific for fructose, mannose, and galactose. No specific kinases were found for gluconate or mannitol, although in the presence of mannitol adenosine triphosphate was utilized by a cytoplasmic fraction. The possibility is discussed that the main pathways of gluconate and mannitol metabolism are oxidation followed by phosphorylation.

Carbohydrate Metabolism↗
PubMed1979

[Rheological properties of a calcium alginate-potato starch hydrolysate-water system].

The rheological properties of liquid solutions and gels of sodium alginate, calcium gluconate and gelling maltodextrin were investigated and compared with the behaviour of pastes and gels of non-degraded potato starch. The dependence of the rheological properties upon the calcium gluconate concentration is extreme. A marked increase in the viscosity of the liquid solutions and maximal values for the hardness and the breaking strength of the gels are obtained at a calcium gluconate/sodium alginate ratio of 0.3. As to breaking strength, elasticity and hardness, the maltodextrin-based gels are many times inferior to the potato starch-based gels. The breaking strength, hardness and elasticity of maltodextrin gels are increased by the addition of sodium alginate and calcium gluconate, which increases their effectiveness when used in foods.

Alginates↗
PubMed1994

A method for obtaining 13C isotopomer populations in 13C-enriched glucose.

13C NMR analysis of 13C-enriched glucose containing multiple isotopomers is hampered by chemical shift similarities of several carbon resonances and by the presence of two anomeric forms. A convenient and quantitative method of enzymatically oxidizing glucose to gluconate in tissue and perfusate extracts is presented. The six carbon resonances of the resulting 13C-enriched gluconate are fully resolved at high pH, thereby allowing a determination of the fractional population of each 13C isotopomer by 13C NMR. The utility of this method is demonstrated using the effluent from an isolated perfused liver containing 13C-enriched glucose produced by hepatic metabolism of sodium [1,2,3-13C3]propionate via the citric acid cycle and gluconeogenesis. An analysis of the gluconate C2 and C5 resonances in this sample showed that pentose phosphate activity was insignificant during this perfusion protocol. As demonstrated, this method provides a means of fully describing 13C isotopomer populations in enriched glucose samples where isotope may be derived from multiple metabolic pathways, thus expanding the scope of experimental design and enrichment strategies.

Animals↗
PubMed1996

Preservation of the non-rectangular cuticular plate/cell axis angle of outer hair cells.

Motile properties of outer hair cells (OHCs) may contribute to sharp tuning and amplification in the mammalian cochlea. Shape changes of isolated OHCs in response to various physical and chemical influences have been investigated intensively. However, determinations of shape may have been influenced by unanticipated effects of preparation and preservation of the OHCs investigated. Thus, in a first step, lengths of freshly isolated OHCs from the guinea pig cochlea were determined using a video-enhancing magnification system. The cuticular plate/cell axis angle (CP/CA angle) was then measured in native cells and under the influence of potassium chloride and potassium gluconate incubation. To show the influence of glutaraldehyde (GA) fixation on the isolated OHCs, fixative-dependent changes on cell length and CP/CA angle were recorded in native and preincubated OHCs. In these experiments, the cell length of vital isolated OHCs was between 41.5 micrometers, in the basal turn, and 103.7 micrometers, in the apical turn. The average CP/CA angle was 106 degrees +/- 4.2 degrees (n = 324 cells, turns 1-4) with no statistically significant differences for the four turns. Under the influence of potassium chloride, cell length was reduced by 8.1%. Potassium gluconate incubation led to a shortening of cell length, followed by a 5.3% increase after 5 min. The CP/CA angle under potassium chloride was decreased (97.0 degrees) and was then increased under the influence of potassium gluconate (110.7 degrees) as a result of cuticular plate tilting. Cell shrinkage after fixation depended on the fixative's osmolarity and on the GA concentration. Increased GA levels amplified cell shrinkage from 34% for hypo-osmolar solutions to 15% in iso-osmolar and 29% in hyperosmolar solutions. The CP/CA angle of native and incubated OHCs was not different from those fixed with GA. The present data provide a rational basis for isolated OHC shape parameters. Moreover, functionally induced changes can be better interpreted when OHCs are influenced by fixatives, as shown in the GA experiments.

Animals↗
PubMed1992

K+ current stimulation by Cl- in the midgut epithelium of tobacco hornworm (Manduca sexta). I. Kinetics and effect of Cl(-)-site-specific agents.

Goblet cells in the midgut epithelium of the tobacco hornworm (Manduca sexta larva, 5th instar) actively secrete K+. This can be measured as short-circuit current (Isc) when the tissue is mounted in an Ussing chamber and bathed in K(+)-rich standard saline containing 32 mmol K+.l-1. Isc depends strictly on basolateral (i.e. haemolymph side) K+ and is therefore termed K+ current, IK. Basolateral, but not apical, chloride, bromide and iodide stimulate IK when compared to the baseline current recorded with gluconate-, nitrate- or thiocyanate-containing salines. So-called "Cl(-)-specific" transport inhibitors (frusemide, 9-anthracene carboxylic acid, diphenylamine carboxylic acid and 4,4'-diisothiocyana-to-stilbene-2,2'-disulphonic acid) reduce IK when added to the basolateral bath, whether Cl- or gluconate is the principal ambient anion. Cl- stimulates IK according to saturation kinetics. The Michaelis-Menten-type, K+ concentration-dependent, saturation of IK is altered in a highly specific manner when gluconate is replaced by Cl-: maximal K+ current, as well as the apparent Michaelis constant, are increased by a factor of 4. Since IK develops in these conditions exclusively via basolateral, Ba(2+)-blockable K+ channels, these results can be understood if it is assumed that haemolymph Cl- interferes with the K+ channel by simultaneously lowering the binding affinity for K+ ions and increasing their subsequent transfer rate across the basolateral goblet cell membrane.

Animals↗
PubMed1978

Effect of temperature on diauxic growth with glucose and organic acids in Pseudomonas fluorescens.

Growth of Pseudomonas fluorescens in batch culture with glucose and organic acids resulted in typical diauxic responses at 30 degrees C but no detectable diauxic lag at 5 degrees C. At 30 degrees C, organic acids were preferentially utilized during the first growth phase. Glucose utilization was delayed until onset of the second growth phase. Systems involved in direct uptake and catabolism of glucose responded in a manner compatible with repression by malate and/or its metabolites and induction by glucose and/or its metabolites. The oxidative non-phosphorylated pathway, through gluconate and 2-ketogluconate (2-KG) as intermediates, was not induced during either growth phase. At 5 degrees C, growth with glucose and organic acids was biphasic but without diauxic lag. Organic acids were preferentially utilized during the first growth phase. Although carbon from glucose was not fully catabolized until onset of the second growth phase, glucose was oxidized to and accumulated extracellularly as gluconate and 2-KG during the first growth phase. No significant repression of glucose-catabolizing enzymes was observed during growth with organic acids in the presence of glucose. However, uptake activities for gluconate and 2-KG did not increase significantly until onset of the second growth phase. Thus, at low temperatures, psychotrophic P. fluorescens oxidized glucose to extracellular 2-KG, while growing on preferred carbon sources. The 2-KG was then catabolized after depletion of the organic acid.

Carboxylic Acids↗
PubMed1977

Energy conservation during nitrate respiration in Paracoccus denitrificans.

P/2e ratios were calculated from anaerobic chemostat cultures of Paracoccus denitrificans with nitrogenous oxides as electron acceptor. P/2e ratios were calculated, using the YmaxATP values determined for aerobic cultures. When succinate was the carbon and energy source the average P/2e values of the sulphate- and succinate-limited cultures with nitrate as electron acceptor were 0.5 and 0.7, respectively, and of the nitrite-limited culture 0.9. With gluconate as carbon and energy source and average P/2e values of the gluconate-limited with nitrate as electron acceptor and nitrate limited cultures were 0.9 and 1.1, respectively. leads to H+/O ratios measured in cells obtained from sulphate-, succinate, nitrite-, gluconate- and nitrate-limited cultures yielded respective average values of 3.4, 4.5, 3.5, 4.8 and 6.2 for endogenous substrates. From our data we conclude that sulphate- and nitrite- limitation causes the loss of site I phosphorylation. Nitrite has no influence on the maximum growth yield on ATP. We propose that metabolism in heterotrophically grown cells of Paracoccus dentrificans is regulated on the level of phosphorylation in the site I region of the electron transport chain.

Anaerobiosis↗
PubMed1986

Direct demonstration of high transepithelial chloride-conductance in normal human sweat duct which is absent in cystic fibrosis.

The transepithelial electrical potential difference (Vt) and the transepithelial resistance (Rt) were measured across individual microdissected sweat ducts obtained from biopsies of the scapular region of normal human volunteers and of a Cystic Fibrosis (CF) patient. During luminal and contraluminal perfusion with NaCl Ringer solution Vt and Rt were -10.7 +/- 1.1 (mV, lumen negative) and 10.9 +/- 1.7 (omega X cm2). Bilateral substitution of Cl- by gluconate increased Vt and Rt to -42.7 +/- 5.1 (mV) and 98.0 +/- 12.0 (omega X cm2), respectively. Luminal application of amiloride (10(-4) mol/l) collapsed Vt and increased Rt to 11.4 +/- 1.7 and 159.7 +/- 12.0 omega X cm2 in Cl- and gluconate Ringer respectively. These data indicate that 90% of the total transepithelial conductance is attributed to Cl- and 5% to Na+ while the remaining 5% is unaccounted for at present. In CF ducts Vt and Rt were high already in Cl- Ringer solution (-78.9 +/- 9.9 mV and 91.2 +/- 15.6 omega X cm2) and did not change significantly after replacement of Cl- by gluconate. The data demonstrate that normal human sweat duct epithelium, despite its ability to generate high electric and osmotic gradients, is a low resistance epithelium, probably because of a high Cl(-)-permeability of its cell membranes. In addition, the data demonstrate that this Cl(-)-conductance is missing in CF, in conformation of what has been postulated previously from simple potential measurements.

Adult↗
PubMed1988

Influence of serosal Cl on transport properties and cation activities in frog skin.

The effects of serosal substitution of isosmotic Na2SO4-Ringer solution for NaCl-Ringer solution were studied in the short-circuited frog skin (Rana pipiens, Northern variety). Despite prompt changes of transepithelial measurements, initial cellular effects were slight. After 30 to 45 min, however, the transcellular current had decreased and the cell electrical potential had depolarized, in association with decrease of the apical membrane fractional resistance and basolateral membrane conductance. Apical membrane slope conductance was unaffected. Similar effects were obtained with isolated epithelia. With the use of gluconate or NO3 in place of Cl, the effects on cellular current and conductance were minimal or insignificant, despite changes of the cell potential, fractional resistance, and basolateral conductance similar to those seen with sulfate. Following prolonged exposure to serosal SO4-Ringer, the extent of depolarization induced by raising the serosal K concentration decreased, indicating diminution of basolateral K conductance and the existence of other basolateral conductances. Equilibration in serosal gluconate-Ringer enhanced polarization on serosal restoration of Cl or removal of Na, again indicating a time-dependent change in the basolateral conductance pattern. Depolarization on removal of serosal Cl was not attributable to inhibition of the pump. Nor was it the result of decrease of the K equilibrium potential EK: exposure to serosal SO4-Ringer decreased cell K activity aKc from 104 +/- 6 to 58 +/- 4 mM (n = 5), but EK was reduced only slightly; exposure to serosal gluconate increased aKc and EK. Serosal sulfate lowered the cell Na activity aNac, but the electrochemical potential difference for Na across the apical surface was unaffected. The concurrent decrease of both aKc and aNac following serosal substitution of SO4 for Cl raises questions concerning mechanisms of osmoregulation.

Amiloride↗
PubMed1987

Basolateral membrane K permselectivity and regulation in bullfrog cornea epithelium.

In the isolated bullfrog cornea epithelium, under short-circuit conditions the regulation of the K permeability of the basolateral membrane was studied with conventional and K-selective microelectrodes in Cl-free Ringers. In Cl-free Ringers, the transcellular current is less than 1 microA/cm2, allowing estimation of the basolateral membrane electromotive force from measurements of the membrane voltage (Vsc). The apparent basolateral membrane K conductance was determined from measurements of the effects of single ion substitutions of K for Na on the Vsc. An increase of K from 2.5 to 25 mM on the stromal side depolarized the membrane voltage by 29 mV, whereas additional increases to 56 and 100 mM resulted in depolarizations consistent with a Nernstian prediction. In the range between 25 and 56 mM K, these decreases in membrane voltage were smaller after either decreasing the stromal-side pH from 8.1 to 7.2 or substitution of sulfate with gluconate. In contrast, preincubation with 0.1 mM ouabain did not change the membrane voltage depolarizations over any of the K ranges between 2.5 and 100 mM. Equivalent circuit analysis, based on the effects of nystatin on the electrical parameters, was used to validate the changes in the apparent basolateral membrane K conductance following increases in [K], substitution of SO4 with gluconate and Na:K pump inhibition. An increase in the [K] to 120 mM decreased the basolateral membrane resistance nearly three-fold, whereas gluconate substitution resulted in a 2.5-fold increase of the basolateral membrane resistance. This resistance increased an additional 2-fold after exposure to 5 mM Ba.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1996

Formation of intracytoplasmic lipid inclusions by Rhodococcus opacus strain PD630.

An oleaginous hydrocarbon-degrading Rhodococcus opacus strain (PD630) was isolated from a soil sample. The cells were able to grow on a variety of substrates and to produce large amounts of three different types of intracellular inclusions during growth on alkanes, phenylalkanes, or non-hydrocarbon substrates. Electron microscopy revealed large numbers of electron-transparent inclusions with a sphere-like structure. In addition, electron-dense inclusions representing polyphosphate and electron-transparent inclusions with an elongated disc-shaped morphology occurred in small amounts. The electron-transparent inclusions of alkane- or gluconate-grown cells were composed of neutral lipids (98%, w/w), phospholipids (1.2%, w/w), and protein (0.8%, w/w). The major component of the cellular inclusions was triacylglycerols; minor amounts of diacylglycerols and probably also some free fatty acids were also present. Free fatty acids and/or fatty acids in acylglycerols in cells of R. opacus amounted up to 76 or 87% of the cellular dry weight in gluconate- or olive-oil-grown cells, respectively. The fatty acid composition of the inclusions depended on the substrate used for cultivation. In cells cultivated on n-alkanes, the composition of the fatty acids was related to the substrate, and intermediates of the beta-oxidation pathway, such as hexadecanoic or pentadecanoic acid, were among the acylglycerols. Hexadecanoic acid was also the major fatty acid (up 36% of total fatty acids) occurring in the lipid inclusions of gluconate-grown cells. This indicated that strain PD630 utilized beta-oxidation and de novo fatty acid biosynthesis for the synthesis of storage lipids. Inclusions isolated from phenyldecane-grown cells contained mainly the non-modified substrate and phenylalkanoic acids derived from the hydrocarbon oxidation, such as phenyldecanoic acid, phenyloctanoic acid, and phenylhexanoic acid, and approximately 5% (w/w) of diacylglycerols. The lipid inclusions seemed to have definite structures, probably with membranes at their surfaces, which allow them to maintain their shape, and with some associated proteins, probably involved in the inclusion formation.

Fatty Acids↗
PubMed2005

Urothelial injury to the rabbit bladder caused by calcium dissolving agents including two new citrate solutions.

We compared the urothelial injury to the bladder caused by four agents capable of dissolving calcium salts. The solutions were administrated in an antegrade way through left ureterostomies in 54 rabbits for periods of 24, 48 and 72 h. The bladders were then removed and three routine histological sections were made for each. The following six solutions were used: physiological sodium chloride solution (Phys), artificial urine (Art), 0.03 M disodium EDTA buffered to pH 8.5 with triethanolamine (EDTA), 10% Renacidin (R), test solution 2 (S2, using D-gluconic acid-lactone and other compounds that differ from R in terms of ingredients or quantity), and test solution 1 (S1, using D-gluconic-acid instead of D-gluconic acid-lactone in S2 but keeping the other ingredients the same) for irrigation. At 24 h there was no observable urothelial damage caused by perfusion with Phys or Art; solutions R, S1 and S2 caused approximately the same level of injury to the rabbit bladder mucosa; however, irrigation with disodium-EDTA caused more serious urothelial injury than R, S1 and S2 (P<0.05, chi2-test) and may be unacceptable. The damage to bladder tissues treated with S1 and S2 was less than that caused by R, but this was not significant (P>0.05, chi2-test). Following a prolonged irrigation time, all of these solutions cause further urothelial damage, but EDTA caused the most, followed by R, S1, S2, Phys or Art, respectively, at 48 and 72 h. In view of the better solubility effect of solutions S1 and S2 compared with R, it might be justified in accepting the more pronounced urothelial irritation caused these solutions, but in order to enhance their effectiveness and reduce urothelial injury further study will be needed.

Animals↗
PubMed2002

Cloning, characterization and comparison of the Pseudomonas mendocina polyhydroxyalkanoate synthases Phac1 and PhaC2.

This study describes a comparison of the polyhydroxyalkanoate (PHA) synthases PhaC1 and PhaC2 of Pseudomonas mendocina. The P mendocina pha gene locus, encoding two PHA synthase genes [phaC1Pm and phaC2pm flanking a PHA depolymerase gene (phaZ)], was cloned, and the nucleotide sequences of phaC1Pm (1,677 bp), phaZ (1,034 bp), and phaC2pm (1,680 bp) were determined. The amino acid sequences deduced from phaC1Pm and phaC2pm showed highest similarities to the corresponding PHA synthases from other pseudomonads sensu stricto. The two PHA synthase genes conferred PHA synthesis to the PHA-negative mutants P. putida GPp104 and Ralstonia eutropha PHB-4. In P. putida GPp 104, phaC1Pm and phaC2Pm mediated PHA synthesis of medium-chain-length hydroxyalkanoates (C6-C12) as often reported for other pseudomonads. In contrast, in R. eutropha PHB-4, either PHA synthase gene also led to the incorporation of 3-hydroxybutyrate (3HB) into PHA. Recombinant strains of R. eutropha PHB-4 harboring either P. mendocina phaC gene even accumulated a homopolyester of 3HB during cultivation with gluconate, with poly(3HB) amounting to more than 80% of the cell dry matter if phaC2 was expressed. Interestingly, recombinant cells harboring the phaC1 synthase gene accumulated higher amounts of PHA when cultivated with fatty acids as sole carbon source, whereas recombinant cells harboring PhaC2 synthase accumulated higher amounts when gluconate was used as carbon source in storage experiments in either host. Furthermore, isogenic phaC1 and phaC2 knock-out mutants of P. mendocina provided evidence that PhaC1 is the major enzyme for PHA synthesis in P. mendocina, whereas PhaC2 contributes to the accumulation of PHA in this bacterium to only a minor extent, and then only when cultivated on gluconate.

Acyltransferases↗