A clearance model of inhaled man-made fibers in rat lungs.
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A clearance model of inhaled man-made fibers (MMFs) was developed, and the calculated fiber numbers and dimensions were compared with the experimental ones using a glass fiber (GF), ceramic fiber (RF1) and two potassium octatitanate whiskers (PT1, TW). If the translocation rate by macrophages is constant and the effect of dissolution and disintegration can be ignored, the fiber number is expected to decrease exponentially with time. In the experimental study, however, the fiber number did not always decrease exponentially. In the case of RF1, the fiber number decreased almost exponentially and the diameter decreased linearly with the time. The clearance rate constant of GF during 3 to 6 months after the end of one-month exposure was greater than that during 1 to 3 months. On the contrary, the clearance rate constants of PT1 and TW during 1 to 6 months were greater than next six months. The diameter and the length of GF did not change significantly. The fiber length of PT1 tends to become longer with time although the diameter did not change significantly. Our theoretical model gives a satisfactory fit to these experimental results. (+info)
Metabolic engineering of poly(3-hydroxyalkanoates): from DNA to plastic.
(2/1075)
Poly(3-hydroxyalkanoates) (PHAs) are a class of microbially produced polyesters that have potential applications as conventional plastics, specifically thermoplastic elastomers. A wealth of biological diversity in PHA formation exists, with at least 100 different PHA constituents and at least five different dedicated PHA biosynthetic pathways. This diversity, in combination with classical microbial physiology and modern molecular biology, has now opened up this area for genetic and metabolic engineering to develop optimal PHA-producing organisms. Commercial processes for PHA production were initially developed by W. R. Grace in the 1960s and later developed by Imperial Chemical Industries, Ltd., in the United Kingdom in the 1970s and 1980s. Since the early 1990s, Metabolix Inc. and Monsanto have been the driving forces behind the commercial exploitation of PHA polymers in the United States. The gram-negative bacterium Ralstonia eutropha, formerly known as Alcaligenes eutrophus, has generally been used as the production organism of choice, and intracellular accumulation of PHA of over 90% of the cell dry weight have been reported. The advent of molecular biological techniques and a developing environmental awareness initiated a renewed scientific interest in PHAs, and the biosynthetic machinery for PHA metabolism has been studied in great detail over the last two decades. Because the structure and monomeric composition of PHAs determine the applications for each type of polymer, a variety of polymers have been synthesized by cofeeding of various substrates or by metabolic engineering of the production organism. Classical microbiology and modern molecular bacterial physiology have been brought together to decipher the intricacies of PHA metabolism both for production purposes and for the unraveling of the natural role of PHAs. This review provides an overview of the different PHA biosynthetic systems and their genetic background, followed by a detailed summation of how this natural diversity is being used to develop commercially attractive, recombinant processes for the large-scale production of PHAs. (+info)
Reduction of cell lysate viscosity during processing of poly(3-hydroxyalkanoates) by chromosomal integration of the staphylococcal nuclease gene in Pseudomonas putida.
(3/1075)
Poly(3-hydroxyalkanoates) (PHAs) are biodegradable thermoplastics which are accumulated by many bacterial species in the form of intracellular granules and which are thought to serve as reserves of carbon and energy. Pseudomonas putida accumulates a polyester, composed of medium-side-chain 3-hydroxyalkanoic acids, which has excellent film-forming properties. Industrial processing of PHA involves purification of the PHA granules from high-cell-density cultures. After the fermentation process, cells are lysed by homogenization and PHA granules are purified by chemical treatment and repeated washings to yield a PHA latex. Unfortunately, the liberation of chromosomal DNA during lysis causes a dramatic increase in viscosity, which is problematic in the subsequent purification steps. Reduction of the viscosity is generally achieved by the supplementation of commercially available nuclease preparations or by heat treatment; however, both procedures add substantial costs to the process. As a solution to this problem, a nuclease-encoding gene from Staphylococcus aureus was integrated into the genomes of several PHA producers. Staphylococcal nuclease is readily expressed in PHA-producing Pseudomonas strains and is directed to the periplasm, and occasionally to the culture medium, without affecting PHA production or strain stability. During downstream processing, the viscosity of the lysate from a nuclease-integrated Pseudomonas strain was reduced to a level similar to that observed for the wild-type strain after treatment with commercial nuclease. The nuclease gene was also functionally integrated into the chromosomes of other PHA producers, including Ralstonia eutropha. (+info)
Production of poly(3-hydroxybutyric acid-co-4-hydroxybutyric acid) and poly(4-hydroxybutyric acid) without subsequent degradation by Hydrogenophaga pseudoflava.
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A Hydrogenophaga pseudoflava strain was able to synthesize poly(3-hydroxybutyric acid-co-4-hydroxybutyric acid) [P(3HB-co-4HB)] having a high level of 4-hydroxybutyric acid monomer unit (4HB) from gamma-butyrolactone. In a two-step process in which the first step involved production of cells containing a minimum amount of poly(3-hydroxybutyric acid) [P(3HB)] and the second step involved polyester accumulation from the lactone, approximately 5 to 10 mol% of the 3-hydroxybutyric acid (3HB) derived from the first-step culture was unavoidably reincorporated into the polymer in the second cultivation step. Reincorporation of the 3HB units produced from degradation of the first-step residual P(3HB) was confirmed by high-resolution 13C nuclear magnetic resonance spectroscopy. In order to synthesize 3HB-free poly(4-hydroxybutyric acid) [P(4HB)] homopolymer, a three-stage cultivation technique was developed by adding a nitrogen addition step, which completely removed the residual P(3HB). The resulting polymer was free of 3HB. However, when the strain was grown on gamma-butyrolactone as the sole carbon source in a synthesis medium, a copolyester of P(3HB-co-4HB) containing 45 mol% 3HB was produced. One-step cultivation on gamma-butyrolactone required a rather long induction time (3 to 4 days). On the basis of the results of an enzymatic study performed with crude extracts, we suggest that the inability of cells to produce 3HB in the multistep culture was due to a low level of 4-hydroxybutyric acid (4HBA) dehydrogenase activity, which resulted in a low level of acetyl coenzyme A. Thus, 3HB formation from gamma-butyrolactone is driven by a high level of 4HBA dehydrogenase activity induced by long exposure to gamma-butyrolactone, as is the case for a one-step culture. In addition, intracellular degradation kinetics studies showed that P(3HB) in cells was completely degraded within 30 h of cultivation after being transferred to a carbon-free mineral medium containing additional ammonium sulfate, while P(3HB-co-4HB) containing 5 mol% 3HB and 95 mol% 4HB was totally inert in interactions with the intracellular depolymerases. Intracellular inertness could be a useful factor for efficient synthesis of the P(4HB) homopolymer and of 4HB-rich P(3HB-co-4HB) by the strain used in this study. (+info)
Analyses of a polyhydroxyalkanoic acid granule-associated 16-kilodalton protein and its putative regulator in the pha locus of Paracoccus denitrificans.
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The polyhydroxyalkanoic acid (PHA) granule-associated 16-kDa protein (GA16 protein) of Paracoccus denitrificans was identified, and its corresponding gene was cloned and analyzed at the molecular level. The N-terminal amino acid sequence of GA16 protein revealed that its structural gene is located downstream from the PHA synthase gene (phaCPd) cloned recently (S. Ueda, T. Yabutani, A. Maehara, and T. Yamane, J. Bacteriol. 178:774-779, 1996). Gene walking around phaCPd revealed two new open reading frames (ORFs) possibly related to PHA synthesis, one of which was the phaPPd gene, encoding GA16 protein, and the other was the phaRPd gene, encoding a protein that is putatively involved in the regulation of the expression of phaPPd. Overproduction of PhaPPd was observed in Escherichia coli carrying phaPPd, but the overproduction was not observed in the presence of phaRPd. Coexpression of phaPPd and PHA biosynthesis genes in E. coli caused increases in both the number of poly-(3-hydroxybutyric acid) (PHB) granules and PHB content and caused decreases in both the size of the granules and the molecular weight of PHB. GA16 protein was considered a phasin protein. The phaRPd gene had significant similarities to stdC, a possible transcriptional factor of Comamonas testosteroni, as well as to other ORFs of unknown function previously found in other PHA-synthetic bacteria. (+info)
Paracrine immunotherapy with interleukin-2 and local chemotherapy is synergistic in the treatment of experimental brain tumors.
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Potent immune responses against malignant brain tumors can be elicited by paracrine intracranial (i.c.) immunotherapy with interleukin (IL)-2. Additionally, i.c. delivery of carmustine via biodegradable polymers has been shown to significantly prolong survival in both animal models and clinical trials. In this study, we show that the combination of paracrine immunotherapy, with nonreplicating genetically engineered tumor cells that produce IL-2, and local delivery of chemotherapy by biodegradable polymers prolongs survival in a synergistic manner in mice challenged intracranially with a lethal murine brain tumor. Animals receiving IL-2-transduced cells and polymers containing 10% 1,3-bis(2-chloroethyl)-1-nitrosourea had significantly improved survival compared with animals receiving IL-2-transduced cells or 10% 1,3-bis(2-chloroethyl)-1-nitrosourea alone. Median survival for the control group was 19 days. Survival in animals receiving IL-2-transduced cells and 1% carboplatin-containing polymers was also significantly improved compared with either therapy alone. Histopathological examination on day 14 of animals receiving combination treatment showed rare degenerating tumor cells. In addition to tissue necrosis surrounding the polymer, a marked inflammatory reaction was observed. In long-term survivors (all animals receiving combination treatment), no tumor was observed and the inflammatory reaction was completely resolved. The brains of animals receiving combination therapy showed both tissue necrosis due to local chemotherapy and strong inflammation due to paracrine immunotherapy. The demonstration of synergy between paracrine IL-2 and local i.c. delivery of antineoplastic drugs is novel and may provide a combined treatment strategy for use against both primary and metastatic i.c. tumors. (+info)
Health hazards in the production and processing of some fibers, resins, and plastics in Bulgaria.
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Results of the toxicological studies of working conditions, general and professional morbidity, and complex examinations carried out on workers engaged in the production of polyamides, polyacrylonitrile fibers, polyester fibers and poly (vinyl chloride) resin, urea-formaldehyde glue, glass fibre materials and polyurethane resins are given. An extremely high occupational hazard for workers in the production of poly (vinyl chloride) resin and porous materials from polyurethane resins and urea-formaldehyde glue has been established. Cases of vinyl chloride disease, poisoning from formaldehyde, isocyanates, and styrene were noted. Prophylactic measures were taken in Bulgaria to lessen the occupational hazard in the productions as set forth included limitation of the work day to 6 hr, free food, additional bonus and leave, and annual physical examinations of workers. (+info)
Removal of endotoxin during purification of poly(3-hydroxybutyrate) from gram-negative bacteria.
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Poly(3-hydroxybutyrate) (PHB) was produced by cultivating several gram-negative bacteria, including Ralstonia eutropha, Alcaligenes latus, and recombinant Escherichia coli. PHB was recovered from these bacteria by two different methods, and the endotoxin levels were determined. When PHB was recovered by the chloroform extraction method, the endotoxin level was less than 10 endotoxin units (EU) per g of PHB irrespective of the bacterial strains employed and the PHB content in the cell. The NaOH digestion method, which was particularly effective for the recovery of PHB from recombinant E. coli, was also examined for endotoxin removal. The endotoxin level present in PHB recovered by 0.2 N NaOH digestion for 1 h at 30 degrees C was higher than 10(4) EU/g of PHB. Increasing the digestion time or NaOH concentration reduced the endotoxin level to less than 1 EU/g of PHB. It was concluded that PHB with a low endotoxin level, which can be used for various biomedical applications, could be produced by chloroform extraction. Furthermore, PHB with a much lower endotoxin level could be produced from recombinant E. coli by simple NaOH digestion. (+info)