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PubMed2001

Control of hemA expression in Rhodobacter sphaeroides 2.4.1: effect of a transposon insertion in the hbdA gene.

The common precursor to all tetrapyrroles is 5-aminolevulinic acid (ALA), and in Rhodobacter sphaeroides its formation occurs via the Shemin pathway. ALA synthase activity is encoded by two differentially regulated genes in R. sphaeroides 2.4.1: hemA and hemT. In our investigations of hemA regulation, we applied transposon mutagenesis under aerobic conditions, followed by a selection that identified transposon insertion mutants in which hemA expression is elevated. One of these mutants has been characterized previously (J. Zeilstra-Ryalls and S. Kaplan, J. Bacteriol. 178:985-993, 1996), and here we describe our analysis of a second mutant strain. The transposon inserted into the coding sequences of hbdA, coding for S-(+)-beta-hydroxybutyryl-coenzyme A dehydrogenase and catalyzing an NAD-dependent reaction. We provide evidence that the hbdA gene product participates in polyhydroxybutyrate (PHB) metabolism and, based on our findings, we discuss possibilities as to how defective PHB metabolism might alter the level of hemA expression.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1996

Marked inflammatory sequelae to implantation of biodegradable and nonbiodegradable polymers in porcine coronary arteries.

BACKGROUND: With the thrombogenic tendency and permanent implant nature of metallic stents, synthetic polymers have been proposed as candidate materials for stents and local drug delivery designs. We investigated the biocompatibility of several synthetic polymers after experimental placement in the coronary artery. METHODS AND RESULTS: Five different biodegradable polymers (polyglycolic acid/polylactic acid [PGLA], polycaprolactone [PCL], polyhydroxybutyrate valerate [PHBV], polyorthoester [POE], and polyethyleneoxide/polybutylene terephthalate [PEO/ PBTP]) and three nonbiodegradable polymers (polyurethane [PUR], silicone [SIL], and polyethylene terephthalate [PETP]) were tested as strips deployed longitudinally across 90 degrees of the circumferential surface of coil wire stents. Appropriately sized polymer-loaded stents were implanted in porcine coronary arteries of 2.5- to 3.0-mm diameter. Four weeks after implantation, stent patency was assessed by angiography followed by microscopic examination of the coronary arteries. The biodegradable PCL, PHBV, and POE and the nonbiodegradable PUR and SIL evoked extensive inflammatory responses and fibrocellular proliferation (thickness of tissue response: 0.79 +/- 0.22, 1.12 +/- 0.01, 2.36 +/- 0.60, 1.24 +/- 0.36, and 1.43 +/- 0.15 mm, respectively). Less but still severe responses were observed for the biodegradable PGLA and PEO/PBTP (0.46 +/- 0.18 and 0.61 +/- 0.23 mm, respectively) and for the nonbiodegradable PETP (0.46 +/- 0.11 mm). CONCLUSIONS: An array of both biodegradable and nonbiodegradable polymers has been demonstrated to induce a marked inflammatory reaction within the coronary artery with subsequent neointimal thickening, which was not expected on the basis of in vitro tests. The observed tissue response may be attributable to a combination of parent polymer compound, biodegradation products, and possibly implant geometry.

Animals↗
PubMed2003

Production of purified polyhydroxyalkanoates (PHAs) for applications in contact with blood.

Samples of olyhydroxyalkanoates (PHAs), polyhydroxybutyrate (PHB) and copolymers poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) with 4 and 18 mol% hydroxyvalerate, synthesized by the bacteria Ralstonia eutropha B5786, were investigated. PHA films in contact with blood did not activate the hemostasis system at the level of cell response, but they did activate the coagulation system and the complement reaction. To detect biologically-active components in the PHAs, a detailed analysis of the composition of the polymers was conducted. Gas chromatography-mass spectrometry revealed long-chain fatty acids (FAs) in the tested PHAs. Their total concentration in the polymer ranged from tenths of mol% to 2-3 mol%, depending on the purification method. C16:0 constituted the largest proportion, up to 70%. Of the long-chain hydroxy acids, only beta-OH-C14:0 was detected and it did not exceed 0.06 mol%. The analysis of the hemocompatibility properties of the PHAs purified by a specialized procedure, including the quantitative and morphological estimation of platelets adherent to the surface of polymer films, the plasma recalcification time and complement activation studies, indicated that PHB and PHBV can be used in contact with blood. It has been found out that the lipopolysaccharides of bacteria producing PHAs, which contain mostly long-chain hydroxy acids, can be the factor activating the hemostasis systems. Thus, the technology of PHA purification must satisfy rather stringent specific requirements.

Biocompatible Materials↗
PubMed2006

A hybrid PHB-hydroxyapatite composite for biomedical application: production, in vitro and in vivo investigation.

Samples of a hybrid composite of polyhydroxybutyrate (PHB), a biodegradable polyester, and hydroxyapatite (HA), with different PHB/HA ratios, have been prepared using mechanical-physical method. Electron microscopy, X-ray structure analysis and differential thermal analysis have been used to investigate the structure and physicochemical properties of the composite, depending on the PHB/HA ratio. The properties of the surface of the HA-loaded composite are significantly different from those of the pure polymer. As the HA percentage in the composite increases, free interface energy, the cohesive force, i.e., the strength of the adhesive bond between the composite surface and the water phase, and surface wettability increase. The HA percentage of the composite does not influence its melting temperature, but affects the temperature for the onset of decomposition: as the HA content increases from 0 to 10% (w/w), Td decreases from 260 degrees C to 225 degrees C. The degree of crystallinity of PHB/HA increases from 77% to 89% with an increase in the HA fraction from 10% to 50%. Functional properties of the composites have been investigated in vitro and in vivo. The best parameters of growth and differentiation of murine marrow osteoblasts are registered on PHB/HA samples containing 10% and 20% HA. In ectopic bone formation assay it has been proven that the hybrid PHB/HA composites can function as scaffolds and that bone tissue develops on their surface and in pores.

Animals↗
PubMed2005

Proteome analysis of a recombinant Bacillus megaterium strain during heterologous production of a glucosyltransferase.

A recombinant B. megaterium strain was used for the heterologous production of a glucosyltransferase (dextransucrase). To better understand the physiological and metabolic responses of the host cell to cultivation and induction conditions, proteomic analysis was carried out by combined use of two-dimensional gel electrophoresis and mass spectrometry (2-DE/MS) for protein separation and identification. 2-DE method was optimized for the separation of intracellular proteins. Since the genome of B. megaterium is not yet available, peptide sequencing using peptide fragment information obtained from nanoelectrospray ionization quadrupole-time-of-flight tandem mass spectrometry (ESI-QqTOF MS/MS) was applied for protein identification. 167 protein spots were identified as 149 individual proteins, including most enzymes involved in the central carbon metabolic pathways and many enzymes related to amino acid synthesis and protein synthesis. Based on the results a 2-DE reference map and a corresponding protein database were constructed for further proteomic approaches on B. megaterium. For the first time it became possible to perform comparative proteomic analysis on B. megaterium in a batch culture grown on glucose with xylose induction for dextrasucrase production. No significant differences were observed in the expression changes of enzymes of the glycolysis and TCA cycle, indicating that dextransucrase production, which amounted to only 2 % of the entire protein production, did not impose notable metabolic or energetic burdens on the central carbon metabolic pathway of the cells. However, a short-term up-regulation of aspartate aminotransferase, an enzyme closely related to dextransucrase production, in the induced culture demonstrated the feasibility to use 2-DE method for monitoring dextransucrase production. It was also observed that under the cultivation conditions used in this study B. megaterium tended to channel acetyl-CoA into pathways of polyhydroxybutyrate production. No expression increases were found with cytosolic chaperones such as GroEL and DnaK during dextransucrase production and secretion, whereas a strong up-regulation of the oligopeptide-binding protein OppA was observed in correlation with an increased secretion of dextransucrase into the culture medium.

Journal Article↗
PubMed2025

A genome-scale metabolic model for the denitrifying bacterium Thauera sp. MZ1T accurately predicts degradation of pollutants and production of polymers.

The denitrifying bacterium Thauera sp. MZ1T, a common member of microbial communities in wastewater treatment facilities, can produce different compounds from a range of carbon (C) and nitrogen (N) sources under aerobic and anaerobic conditions. In these different conditions, Thauera modifies its metabolism to produce different compounds that influence the microbial community. In particular, Thauera sp. MZ1T produces different exopolysaccharides with floc-forming properties, impacting the physical disposition of wastewater consortia and the efficiency of nutrient assimilation by the microbial community. Under N-limiting conditions, Thauera sp. MZ1T decreases its growth rate and accelerates the accumulation of polyhydroxyalkanoate-related (PHA) compounds including polyhydroxybutyrate (PHB), which plays a fundamental role as C and energy storage in this β-proteobacterium. However, the metabolic mechanisms employed by Thauera sp. MZ1T to assimilate and catabolize many of the different C and N sources under aerobic and anaerobic conditions remain unknown. Systems biology approaches such as genome-scale metabolic modeling have been successfully used to unveil complex metabolic mechanisms for various microorganisms. Here, we developed a comprehensive metabolic model (M-model) for Thauera sp. MZ1T (iThauera861), consisting of 1,744 metabolites, 2,384 reactions, and 861 genes. We validated the model experimentally using over 70 different C and N sources under both aerobic and anaerobic conditions. iThauera861 achieved a prediction accuracy of 95% for growth on various C and N sources and close to 85% for assimilation of aromatic compounds under denitrifying conditions. The M-model was subsequently deployed to determine the effects of substrates, oxygen presence, and the C:N ratio on the production of PHB and exopolysaccharides (EPS), showing the highest polymer yields are achieved with nucleotides and amino acids under aerobic conditions. This comprehensive M-model will help reveal the metabolic processes by which this ubiquitous species influences communities in wastewater treatment systems and natural environments.

Thauera↗
PubMed1990

FSH causes a time-dependent stimulation of preovulatory follicle growth in the absence of pulsatile LH secretion in ewes chronically treated with gonadotrophin-releasing hormone agonist.

The study investigated the relationship between the plasma concentration of FSH and the stimulation of preovulatory follicle growth in vivo in ewes chronically treated with the gonadotrophin-releasing hormone (GnRH) agonist buserelin (HOE 766). Welsh Mountain ewes with regular oestrous cycles were treated for 6 weeks with two discs implants placed s.c., each containing 5 mg of the agonist in a matrix of polyhydroxybutyric acid. Treatment with the agonist for 35 days produced a sustained suppression of the plasma concentration of FSH, stopped the pulsatile release of LH and prevented follicular development beyond 2.5 mm diameter. There was no difference between the total number of follicles greater than 1.0 mm diameter present in the ovaries of GnRH agonist-treated ewes and day 8 luteal phase control ewes. During the sixth week of agonist treatment ewes were infused with ovine FSH (6 micrograms NIADDK-oFSH-16/h) in the presence of only basal concentrations of LH. After 24, 48, 72 or 120 h of FSH infusion, the mean number of follicles greater than 1.0 mm diameters per ewe was not significantly different between treated and control animals. Infusion of FSH caused a time-dependent increase in (1) the number of follicles per ovary greater than 2.5 mm, (2) the mean diameter of these follicles and (3) the proportion of the large follicles which could be classified as oestrogenic (greater than 3.7 nmol oestradiol/follicle per h in vitro). Injection of human chorionic gonadotrophin (750 IU i.m.) after 120 h of FSH infusion caused the majority of these large follicles to ovulate and form apparently normal corpora lutea. These results indicate that, in the absence of pulsatile LH, FSH stimulates the growth of normal large oestrogenic follicles which, when stimulated, ovulate to produce viable corpora lutea.

Animals↗
PubMed2006

Microbial community analysis with a high PHA storage capacity.

Activated sludge was submitted to aerobic dynamic substrate feeding for the production of biodegradable plastics. Two sequencing batch reactors were operated with acetate or propionate as sole carbon substrates. With acetate a homopolymer of polyhydroxybutyrate (PHB) was obtained and with propionate a copolymer of hydroxybutyrate and hydroxyvalerate P(HB/HV) was produced. Three main morphotypes were identified in both sludges: two belong to the Alphaproteobacteria class and the third to the Betaproteobacteria class. Bacilli belonging to Betaproteobacteria were shown by FISH analysis, applied in combination with Nile Blue post-staining, to be the main responsible for PHAs storage. The latter were affiliated to Azoarcus genus within Betaproteobacteria.

Alkanes↗
PubMed1992

Urethral reconstruction with a new synthetic absorbable device. An experimental study.

Four centimeters of the canine urethra was replaced by a graft consisting of a polyglactin fiber mesh tube, coated with polyhydroxybutyric acid. Microscopic examination 8-12 months later showed almost complete regeneration of the urethral epithelium and the adjacent connective tissue. The neo-urethra was patent and there were no anastomotic strictures, nor was there any inflammatory reaction around the urethra. The validity of this concept in humans remains to be proved.

Animals↗
PubMed1979

Electron microscopy of malachite green--glutaraldehyde fixed bacteria.

Malachite green combined with glutaraldehyde has been used recently as a fixative for preserving and revaling lipid complexes in thin sections of eukaryotic cells examined by electron microscopy. When bacteria were prefixed with the above mixture granular electron dense inclusions were revealed in all cultures tested. These inclusions were replaced by electron transparent areas in cells fixed with glutaraldehyde alone. The structures were frequently located near to or within the nucleoid and adjacent to the cell membrane in Gram-negative bacteria and were associated with the nucleoid and mesosomes in Gram-positive species. Polyhydroxybutyrate granules, generally poorly preserved in thin sections of Aquaspirillum serpens, were well preserved by the malachite green-glutaraldehyde fixative. Malachite green complexes were observed outside of the cells in all preparations. Capsules were neither preserved nor stained.

Aldehydes↗
PubMed1992

A new biodegradable patch for closure of atrial septal defect. An experimental study.

Biodegradable patches made from polyhydroxybutyrate (PHB) were used to close experimentally induced atrial septal defect in six calves. The implants were evaluated after 3-12 months macroscopically and by light and scanning electron microscopy with regard to regeneration of tissue and degradation of the polymer. At implant degradation, complete endothelial layers facing the right and left atrium were observed, with a subendothelial layer of collagen and some smooth-muscle cells. The patch was degraded by polynucleated macrophages, and 12 months postoperatively no polymer material was identifiable at ordinary light microscopy, but in polarized light small particles of polymer with persisting foreign body reaction were still seen. Scanning electron microscopy revealed a complete layer of surface cells with morphologic signs of endothelium. There was no shunt or sign of infection. Biodegradable PHB patches implanted in atrial septal defects in this experimental model thus prompted formation of regenerated tissue that macroscopically and microscopically resembled native atrial septal wall. The total degradation time exceeded 12 months.

Animals↗
PubMed1992

Regeneration of pericardial tissue on absorbable polymer patches implanted into the pericardial sac. An immunohistochemical, ultrastructural and biochemical study in the sheep.

A new absorbable polymer prepared from polyhydroxybutyrate (PHB) was inserted as a pericardial patch in sheep to serve as a temporary scaffold for regeneration of pericardial tissue. Postoperative adhesions were rare or absent. The present study focuses on characterization of the regenerated surface cells. The luminal surface of the regenerated tissue was covered with a complete layer of mesothelium-like cells which at light and scanning electron microscopy resembled those in native pericardium. Immunohistochemical stainings for cytokeratin and thrombomodulin were positive in these cells. Heparan sulfate proteoglycan was found in a basement-membrane-like structure beneath the surface cells, as in the normal pericardium. Transmission electron microscopy of the regenerated surface revealed cells with the characteristics of mesothelium. Prostacyclin production in the regenerated tissue was similar to that in native pericardium. The results indicate regeneration of a mesothelial layer with many of the important functions of native mesothelial cells. This may explain the presently and previously observed prevention of pericardial adhesions after cardiac surgery in this field. Clinical testing of PHB patches as pericardial substitutes is warranted in cardiac surgery when pericardial closure is desired.

Animals↗
PubMed1984

Electro-biorheology.

Studies on piezoelectricity in biopolymers are reviewed with some examples. By measuring anisotropy of elastic and piezoelectric constants in fish skin, the direction of preferred orientation of collagen fibers is determined. Temperature and moisture dependence of real and imaginary parts of the piezoelectric constant observed for oriented film of fibrin gel indicates that temperature-time equivalence holds for the piezoelectric relaxation. The piezoelectric constant for oriented film of polyhydroxybutyrate decreases above glass transition temperature, which is related to the elastic and dielectric relaxations. When alpha-helical molecules of poly-gamma-methyl-L-glutamate are electrically oriented in ethylene dichloride solution and shear stress is given by ultrasound, the piezoelectric polarization is observed. When the film of poly-gamma-methyl-glutamate is wrapped around the femur of rat, the formation of new bone is produced.

Animals↗
PubMedDate not supplied

[Study of the dielectric permeability in the superhigh frequency range of a degraded polyoxybutyrate biopolymer].

The dielectric permeability of the degradable biopolymer polyhydroxybutyrate synthesized by hydrogen-oxidizing bacteria Alcaligenes eutrophus was investigated by the resonance method using original highly sensitive microstrip sensors. For the first time, a linear growth of dielectric permeability (delta epsilon/delta T = 7 x 10(-4) degree-1) due to the flexibility of the polymer chain in the temperature range from 10 to 70 degrees C was revealed. The energy of a bend of the nearest fragments was evaluated (E = 392 K), and its correspondence to the energies of bends of the alcyl groups of low-molecular substances like liquid crystals was established. It was shown that at low values of dielectric permeability in the high-frequency range (epsilon' = 2.25 +/- 0.02), which are stable, in a wide range of frequencies of the electromagnetic field (1 MHz - 1 Hz), polyoxybutyrate can be used in the microwave equipment.

Alcaligenes↗
PubMed2002

Effect of different carbon sources on aerobic storage by activated sludge.

A study of substrate removal by real activated sludge with several synthetic substrates (acetate, ethanol, glutamic acid) and wastewater (raw and filtered) was carried out. Substrate, stored compounds (polyhydroxyalkanoates, PHA and internal carbohydrates), ammonia and oxygen uptake rate (OUR) were analytically determined. Polyhydroxybutyrate (PHB) was stored when the substrate was acetate or ethanol, while no appreciable formation of storage compound was detected using glutamic acid. A low amount of PHB was also formed in tests with raw and filtered wastewater which was probably mainly due to its acetate content. As far as the sum of storage and growth (indirectly estimated through ammonia consumption) did not match the overall solids formation, other unidentified mechanisms of substrate removal were likely to occur (biosorption, accumulation and/or storage of unidentified compounds). ASM3 and two derived models were used in the interpretation of experimental data with reference to synthetic substrates. With reference to synthetic substrates ASM3 can well describe the experimental data only assuming a stored product formation much higher than the analytically detected one, whereas the model that assumes a parallel growth and storage on the substrate can well describe the observed stored product profile only assuming a direct contribution of growth much higher than estimated from ammonia consumption. The model that assumes an accumulation/biosorption stage as first step of substrate removal can better describe the whole experimentally observed behaviour. However as well as in ASM3 this implies that some fraction of removed COD is still unidentified. With reference to real wastewater where the different phenomena were mixed up due to the presence of several substrates, the different models gave similar results.

Absorption↗
PubMedDate not supplied

[Dynamics of activity of the key enzymes of polyhydroxyalkanoate metabolism in Ralstonia eutropha].

The dynamics of accumulation of polyhydroxybutyrate (PHB) and the activities of the key enzymes of PHB metabolism (beta-ketothiolase, acetoacetyl-CoA reductase, PHA synthase, D-hydroxybutyrate dehydrogenase, and PHA depolymerase) in the hydrogen bacterium Ralstonia eutropha B5786 were studied under various conditions of carbon nutrition and substrate availability. The highest activities of beta-ketothiolase, acetoacetyl-CoA reductase, and PHA synthase were recorded at the stage of acceleration of PHB synthesis. The activities of enzymes catalyzing PHB depolymerization (PHB depolymerase and D-hydroxybutyrate dehydrogenase) were low, being expressed only at stimulated endogenous PHB degradation. The change of carbon source (CO2 or fructose) did not cause any marked changes in the time course of enzyme activity.

Acetyl-CoA C-Acyltransferase↗
PubMed1989

In vitro and in vivo evaluation of a bondable compact for the prolonged delivery of triamcinolone acetonide to the oral cavity in patients with lichen planus.

Compacts weighing 40 mg and containing triamcinolone acetonide 70-90% and polyhydroxybutyric acid (PHB) 30-10% or poly (DL-lactic acid) 20% with a diameter of 5 mm were bonded onto the side-wall of molar teeth. In vitro dissolution studies showed the compacts to release 12% of drug in 30 days with an initial burst effect. Drug loading or polymer matrix type had little effect. In vivo studies in dogs showed that compacts containing 80% drug in PHB produced salivary levels of triamcinolone acetonide for 30 days. When evaluated in five patients with lichen planus resistant to conventional therapy, these compacts produce a slight clinical improvement in three subjects. Differential scanning calorimetry studies confirmed that the drug and polymer were present as a physical mix in these compacts.

Adolescent↗
PubMedDate not supplied

[Use of rifampicin for directed modification of the nucleotide composition of the cells of Bacillus megaterium and Candida utilis].

The object of this work was to study the effect of rifampicin on the physiological state of Bacillus megaterium and Candida utilis in chemostat and batch cultures. When B. megaterium is cultivated in a medium containing 1 microgram of rifampicin per litre, its metabolism changes, the pool of free nucleotides rises, and the content of RNA drops in response to the specific action of the inhibitor. The modified state is observed throughout many generations. The action of the inhibitor is also non-specific: protein content falls and polyhydroxybutyric acid is accumulated. The morphology of B. megaterium cells changes: they become larger, their cell wall thickens, and the separation of cells from one another is complicated. At a high concentration (5 mg/l), rifampicin decelerates the growth of C. utilis and causes merely a minor non-specific inhibition.

Bacillus megaterium↗