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It has been reported that Poly-β-hydroxybutyrate (PHB) is generated from acetate in the rice root. However, no information is available about the biosynthetic pathway of PHB from acetate in plant cells. In the bacterium Ralstonia eutropha H16 (R. eutropha), PHB is synthesized from acetyl CoA by the consecutive reaction of three enzymes: β-ketothiolase (EC: 2.3.1.9), acetoacetyl CoA reductase (EC: 1.1.1.36) and PHB synthase (EC: 2.3.1.-). Thus, in this study, we examined whether the above three enzymatic activities were also detected in rice seedlings. The results clearly showed that the activities of the above three enzymes were all detected in rice. In particular, the PHB synthase activity was detected specifically in the sonicated particulate fractions (2000g 10min precipitate (ppt) and the 8000g 30min ppt) of rice roots and leaves. In addition to these enzyme activities, several new experimental results were obtained on PHB synthesis in higher plants: (a) (14)C-PHB generated from 2-(14)C-acetate was mainly localized in the 2000g 10min ppt and the 8000g 30min ppt of rice root. (b) Addition of acetate (0.1-10mM) to culture medium of rice seedlings did not increase the content of PHB in the rice root or leaf. (c) In addition to C3 plants, PHB was generated from acetate in a C4 plant (corn) and in a CAM plant (Bryophyllum pinnatum). d) Washing with ethylenediaminetetraacetic acid (EDTA) strongly suggested that the PHB synthesized from acetate was of plant origin and was not bacterial contamination.

Intraosseous lymphoma in the maxilla or mandible is sometimes difficult to diagnose because of the absence of specific signs and symptoms. This study reports five cases of intraosseous lymphoma in the oral and maxillofacial regions. There were four cases of diffuse large B-cell lymphoma (DLBCL), including EBV-positive DLBCL of the elderly, and one of Burkitt's lymphoma. Regarding the tumor sites, three of the cases were in the maxilla, while two were in the mandible. Two of the conditions were clinically diagnosed as inflammatory diseases at the time of their first examination. Two patients died of their lymphoma during the follow-up period. Based on these events and findings, we clearly realized that careful examination is necessary to be diagnosed early, which results in a much better prognosis. Moreover, maxillofacial surgeons need to keep following up carefully on their patients to immediately recognize any recurrences after completion of treatment.

Incidences of lymphoproliferative disorders (LPDs) in patients with compromised immune systems associated with immunosuppressants such as methotrexate (MTX) administered for the treatment of rheumatoid arthritis (RA) are reportedly increasing. Although extranodal lesions develop in half of the patients with MTX-associated LPDs, only a few studies have reported on intraoral lesions. We evaluated 2 elderly women with MTX-associated LPDs who had received MTX for the treatment of RA and presented with atypical ulceration of the tongue. Biopsy specimens demonstrated polymorphous B-cell LPD, probably associated with MTX. Epstein-Barr virus (EBV) was identified by immunohistochemistry for latent membrane protein 1 and by EBV-encoded RNA in situ hybridization. After MTX withdrawal, in both cases, ulcers showed complete regression at 8 weeks, and no subsequent treatment was required. Close monitoring of LPDs is mandatory, because recurrence within 10 months has been reported in half of the patients in whom LPDs had initially regressed.

We previously reported that the activities of two 3-hydroxybutyrate dehydrogenases (BDH1 and BDH2) were greatly influenced by culture conditions when Ralstonia pickettii T1, a strain growing on extracellular poly-3-hydroxybutyrate (PHB), was grown on different carbon sources such as 3HB and succinate. In this study, knockout mutants of bdh1 or bdh2 were constructed and characterized under different culture conditions. In addition, a novel BDH (BDH3) was found in bdh2 mutants, and bdh3 was cloned. Apparent kinetic parameters for the substrates of BDH3 indicated that the enzyme is suitable for the oxidation reaction of 3-hydroxybutyrate (3HB) to acetoacetate. In Western blotting, it was clear that BDH3 is produced only in cells grown on 3HB or PHB as a carbon source, while BDH1 and BDH2 are produced in cells grown on various carbon sources such as sugars, amino acids, organic acids, 3HB, and PHB. Both the bdh1 and bdh2 mutants lagged behind the wild type in growth rates when the cells were cultured with 3HB, citrate, succinate, or nutrient broth. A test of sensitivity to diamide as an oxidative stress revealed that the lack of BDH1 or BDH2 caused a decline in the capacity to neutralize the stress. These results suggested that BDH1 and BDH2 are needed to regulate the cytoplasmic redox state as well as to utilize 3HB, while BDH3 is specialized to utilize 3HB. The expression of bdh3 may be coordinately regulated with a gene encoding putative 3HB permease.

The extracellular poly(3-hydroxybutyrate) depolymerase from Ralstonia pickettii T1 has been purified, its function and character investigated in detail, and its gene cloned and sequenced. However, the mechanism by which this enzyme is secreted has not been elucidated. A mutant unable to degrade poly(3-hydroxybutyrate), N17, was obtained with the random insertion of a mini-transposon, Tn5. Western analysis using antiserum against the poly(3-hydroxybutyrate) depolymerase of Ralstonia pickettii T1, revealed that N17 accumulated the poly(3-hydroxybutyrate) depolymerase in the periplasm and cytoplasm, and did not secrete the enzyme into the external medium. It was also found that 3-hydroxybutyrate-oligomer hydrolase was secreted but inactive. The disrupted gene in N17, depO, was analyzed by Southern hybridization and its nucleotide sequence was determined. One complete open reading frame was found in the cloned 2.3-kbp DNA fragment. From a BLAST search, this gene product was found to be homologous to PulO of Ralstonia eutropha JMP134 (60% identity) and XcpA of Pseudomonas aeruginosa (60% identity). These proteins are prepilin peptidase/N-metyltransferases, a component of the Type II secretion pathway. DepO also had the four cysteines highly conserved in most prepilin peptidases at the same positions. The transcript of depO was examined by Northern hybridization using depO as a probe. In the total RNA of Ralstonia pickettii T1 in the early stationary phase, a band at 2.6-kb was detected, suggesting depO to be a functional gene. In this study, it was found that poly(3-hydroxybutyrate) depolymerase was secreted by the Type II pathway.

The recently finished genome sequence of Ralstonia eutropha H16 harbors nine genes that are thought to encode functions for intracellular depolymerization (mobilization) of storage poly(3-hydroxybutyrate) (PHB). Based on amino acid similarities, the gene products belong to four classes (PhaZa1 to PhaZa5, PhaZb, PhaZc, and PhaZd1/PhaZd2). However, convincing direct evidence for the in vivo roles of the gene products is poor. In this study, we selected four candidate genes ( phaZa1 , phaZb , phaZc , and phaZd1 ) representing the four classes and investigated the physiological function of the gene products (i) with recombinant Escherichia coli strains and (ii) with R. eutropha null mutants. Evidence for weak but significant PHB depolymerase activity was obtained only for PhaZa1. The physiological roles of the other potential PHB depolymerases remain uncertain.

Poly(3-hydroxybutyrate) (PHB) granules isolated in native form (nPHB granules) from Ralstonia eutropha catalyzed formation of PHB from 14 C-labeled acetyl coenzyme A (CoA) in the presence of NADPH and concomitantly released CoA, revealing that PHB biosynthetic proteins (acetoacetyl-CoA thiolase, acetoacetyl-CoA reductase, and PHB synthase) are present and active in isolated nPHB granules in vitro. nPHB granules also catalyzed thiolytic cleavage of PHB in the presence of added CoA, resulting in synthesis of 3-hydroxybutyryl-CoA (3HB-CoA) from PHB. Synthesis of 3HB-CoA was also shown by incubation of artificial (protein-free) PHB with CoA and PhaZa1, confirming that PhaZa1 is a PHB depolymerase catalyzing the thiolysis reaction. Acetyl-CoA was the major product detectable after incubation of nPHB granules in the presence of NAD + , indicating that downstream mobilizing enzyme activities were also present and active in isolated nPHB granules. We propose that intracellular concentrations of key metabolites (CoA, acetyl-CoA, 3HB-CoA, NAD + /NADH) determine whether a cell accumulates or degrades PHB. Since the degradation product of PHB is 3HB-CoA, the cells do not waste energy by synthesis and degradation of PHB. Thus, our results explain the frequent finding of simultaneous synthesis and breakdown of PHB.

A cDNA encoding Penicillium funiculosum P(3HB) depolymerase (PhaZPfu) was cloned from a cDNA library. This cDNA contained a 1,020-bp open reading frame (ORF) that encoded 339 amino acids. Edman degradation of PhaZPfu indicated that 20 amino acids from the N terminus function as a signal peptide. Homology analysis revealed that PhaZPfu lacks linker and substrate-binding domains, both of which are observed in bacterial P(3HB) depolymerases. This may account for a weak binding affinity of PhaZPfu to the P(3HB) surface.

Fibrous dysplasia of bone is a benign disease characterized by the progressive replacement of normal bone elements by fibrous tissue. The disease can involve any bone in the body. However, involvement of the zygomatic arch is relatively rare. Moreover, depending on the site and extent, fibrous dysplasia may be accompanied by functional disorder. We report a case of fibrous dysplasia of the zygomatic arch that extended to the pterygoid fossa, producing trismus.A 56-year-old woman was referred to our hospital because of a painless swelling in the left cheek and trismus. Computed tomography and simple X-ray examinations of lesion revealed a ground-glass appearance. Biopsy was performed, leading to a suspected diagnosis of osteoma. The lesion interfered with the movement of the mandible. The lesion was resected under general anesthesia. The histopathological diagnosis was fibrous dysplasia. The postoperative course was uneventful, and the lesion has not recurred for 6 years.

We describe a case of acute suppurative arthritis of the temporomandibular joint that gradually progressedto degenerative bony changes of the condyle over a period of 3 years 3months safter treatment. However, the patient remained clinically asymptomatic.The patient, a 51-year-old man, presented with trismus and severe TMJ pain. On initial examination, themandible was found to have shifted to the contralateral side, with malocculusion. The range of motion was limitedto 24mm. CT scans revealed massive swelling of the lateral pterygoid muscle. The right mandibular condylarhead was displaced anteriorly.The patient was admitted to the hospital and underwent surgical drainage from the joint space. The right maxillarymolar stumps were removed. The symptoms completely resolved within 2 weeks. The occlusion returned towithin normal limits. Maximum opening increased to 44mm. However, CT scans revealed worm-hole-like boneresorption in the right TMJ condyle. The patient was discharged from the hospital on 17th day.Follow-up was uneventful, and maximum opening increased to 52mm. CT scans disclosed extensive resorptionof the lateral condyle and proliferative bony changes of the articular surfaces. However, the patient was asymptomaticwithout further disturbance of daily activities, maintaining a range of motion of 48mm.

The enzymatic hydrolysis behavior of compositionally well-fractionated bacterial poly(3-hydroxybutyrate- co -3-hydroxyvalerate) [P(3HB- co -3HV)] with 3HV-unit content from 8 to 98 mol% and bacterial poly(3-hydroxybutyrate) [P(3HB)] has been investigated in the presence of poly(3-hydroxyalkanoate) depolymerases isolated from Ralstonia pickettii T1 and Acidovorax sp. TP4. The water-soluble degradation products were characterized by HPLC and 1 H NMR spectroscopy. P(3HB- co -3HV)s with the whole range of 3HV-unit content can be degraded by R. pickettii T1 depolymerase, while only P(3HB- co -3HV)s with 3HV-unit content less than 80 mol% can be degraded by Acidovorax sp. TP4 depolymerase. It was found that the enzymatic degradation behavior of fractionated P(3HB- co -3HV)s was affected not only by the comonomer unit composition and its distribution but also by the solid-state structure of P(3HB- co -3HV)s and further by the bacterial source of depolymerases. Based on the results, the mechanism of enzymatic degradation was discussed.

The dependences of the phase structure and biodegradability on the thermal histories were investigated for amorphous atactic poly(R,S-3-hydroxybutyrate) (aPHB) in amorphous blends with atactic poly(methyl methacrylate) (PMMA).Poly(R-3-bydroxybutyrate)(PHB) depolymerase isolated from Ralstonia pickettii T1 was used as an enzyme. Pure aPHB in a rubbery state could not be degraded by PHB depolymerase. The results of DSC and TEM observations revealed that aPHB/PMMA blends were partially miscible with compositional heterogeneity. When the blends have a proper phase distribution of both the hydrolyzable rubbery phase and the depolymerase-adsorbable glassy phase, resulting from compositional heterogeneity, the enzymatic degradation of aPHB can be induced by blending with amorphous PMMA. Through the thermal treatment, the weight loss due to enzymatic degradation much increased for some blends.The reason is because such blends formed “micro-separated” structure, that is, the blends had “optimal” phase distributions consisting of enzyme-adsorbable PMMA-rich domains dispersed in the aPHB-rich matrix compositionally closer to pure aPHB. It was suggested that the aPHB component should be degraded faster in the aPHB/PMMA blends with compositional heterogeneity than in the miscible blends.

The enzymatic degradability of chemosynthesized atactic poly([R,S]-3-hydroxybutyrate) [a-P(3HB)] by two types of extracellular poly(3-hydroxyalkanoate) (PHA) depolymerases purified from Ralstonia pickettii T1 (PhaZ(ral)) and Acidovorax Sp. TP4 (PhaZ(aci)), defined respectively as PHA depolymerase types I and II according to the position of the lipase box in the catalytic domain, were studied. The enzymatic degradation of a-P(3HB) by PhaZ(aci) depolymerase was confirmed from the results of weight loss and the scanning electron micrographs. The degradation products were characterized by one- and two-dimension (1)H NMR spectroscopy. It was found that a-P(3HB) could be degraded into monomer, dimer, and trimer by PhaZ(aci) depolymerase at temperatures ranging from 4 to 20 degrees C, while a-P(3HB) could hardly be hydrolyzed by PhaZ(ral) depolymerase in the same temperature range. These results suggested that the chemosynthesized a-P(3HB) could be degraded in the pure state by natural PHA depolymerase.

Poly-3-hydroxybutyrate (PHB) granules of Zoogloea ramigera I-16-M contained two major PHB granule-associated proteins (PGA12 and PGA16) as revealed by sodium dodecyl sulfate-polyacrylamide gel elecrophoresis. N-terminal amino acid sequences of these proteins were determined. The genes encoding these proteins were cloned and sequenced. The structural genes of PGA12 and PGA16 were 351 and 447 bp long, which encode polypeptides with deduced molecular masses of 12.3 and 16.0 kDa, respectively. PGA12 and PGA16 were expressed in Escherichia coli. PHB granules were isolated from cells of recombinant strains of E. coli JM109, which harbored and expressed the PHB-synthetic genes of Ralstonia eutropha H16 and PGA12 or PGA16. These PHB granules contained PGA12 or PGA16 as a major protein. The presence of pga12 or pga16 did not affect the amount of PHB synthesized in E. coli. PGA12 and PGA16 bound to crystalline and amorphous PHB granules.

Polymorphous low-grade adenocarcinoma (PLGA) is a rare malignant tumor arising from the minor salivary glands. We report a case of PLGA occurring in the soft palate. An 82-year-old man had been aware of a soft-palate mass for 7years. The mass was relatively large, measuring 50×60mm, and caused dysphagia and dysphonia. The lesion was clinically diagnosed as a tumor, and the patient underwent resection under general anesthesia. The mass was pathologically diagnosed as PLGA. The postoperative course was uneventful. There has been no recurrence or distant metastasis for 4years.

A poly(3-hydroxybutyrate) (PHB) depolymerase was purified from a fungus, Penicillium funiculosum (IFO6345), with phenyl-Toyopearl and its properties were compared with those of other PHB depolymerases. The molecular mass of the purified enzyme was estimated at about 33 kDa by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The pH optimum and pI were 6.5 and 6.5, respectively. The purified protein showed affinity to Con A-Sepharose, indicating that it is a glycoprotein. Diisopropylfluorophosphate and dithiothreitol inhibited the depolymerase activity completely. The N-terminal amino acid sequence of the purified enzyme was TALPAFNVNPNSVSVSGLSSGGYMAAQL, which contained a “lipase box” sequence. This purified enzyme is one of the extracellular PHB depolymerase which belong to serine esterase. The purified enzyme showed relatively strong hydrolytic activity against 3-hydroxybutyrate oligomers compared with its PHB-degrading activity. PHB-binding experiments showed that P. funiculosum depolymerase has the weakest affinity for PHB of all the depolymerases examined.

To determine the properties of enzymes from bacteria that degrade polypropiolactone (PPL), we isolated 13 PPL-degrading bacteria from pond water, river water, and soil. Nine of these strains were identified as Acidovorax sp., three as Variovorax paradoxus, and one as Sphingomonas paucimobilis. All the isolates also degraded poly(3-hydroxybutyrate) (PHB). A PPL-degrading enzyme was purified to electrophoretical homogeneity from one of these bacteria, designated Acidovorax sp. TP4. The purified enzyme also degraded PHB. The molecular weight of the enzyme was estimated as about 50,000. The enzyme activity was inhibited by diisopropylfluorophosphate, dithiothreitol, and Triton X-100. The structural gene of the depolymerase was cloned in Escherichia coli. The nucleotide sequence of the cloned DNA fragment contained an open reading frame (1476 bp) specifying a protein with a deduced molecular weight of 50,961 (491 amino acids). The deduced overall sequence was very similar to that of a PHB depolymerase of Comamonas acidovorans YM1609. From these results it was concluded that the isolated PPL-degrading enzyme belongs to the class of PHB depolymerases. A conserved amino acid sequence, Gly-X1-Ser-X2-Gly (lipase box), was found at the N-terminal side of the amino acid sequence. Site-directed mutagenesis of the TP4 enzyme confirmed that 20Ser in the lipase box was essential for the enzyme activity. This is the first report of the isolation a PHB depolymerase from Acidovorax.

An intracellular 3-hydroxybutyrate-oligomer hydrolase was purified from a poly(3-hydroxybutyrate)-degrading bacterium, Paucimonas lemoignei. It hydrolyzed the 3-hydroxybutyrate dimer with the highest specific activity of any of the enzymes reported so far. The gene was cloned and sequenced. The deduced amino acid sequence showed that the enzyme is a homolog of the PhaZc of Ralstonia eutropha H16.

Poly(3-hydroxybutyrate) (PHB) depolymerase from Alcaligenes faecalis T1 is composed of three domains: the catalytic (C) domain, the fibronectin type III-like (F) domain, and the substrate-binding (S) domain. We constructed domain deletion, inversion, chimera, and extra-F-domain mutants and examined their enzyme activity and PHB-binding ability. In addition, we performed substitution of 214Asp and 273His with glycine and aspartate, respectively, to examine their participation in a catalytic triad together with 139Ser. The mutant with both the F and S domains deleted and the trypsin-digested enzyme showed no PHB-hydrolyzing activity and less PHB-binding ability than that of the wild-type enzyme but retained D-(-)-3-hydroxybutyrate trimer-hydrolyzing activity at a level similar to that of the wild-type enzyme. The mutant with the F domain deleted and the mutant which had the order of the F and S domains inverted retained PHB-binding ability and trimer-hydrolyzing activity at levels similar to those of the wild-type enzyme but lost PHB-hydrolyzing activity. The chimera mutant, in which the F domain was substituted with a Thr-rich domain of PHB depolymerase A from Pseudomonas lemoignei, and the extra-F-domain mutant, with an additional F domain, retained trimer- and PHB-hydrolyzing activities and PHB-binding ability at levels similar to those of the wild-type enzyme. Two mutants (D214G and H273D) showed no enzymatic activity toward trimer and PHB, and they were not labeled with [3H]diisopropylfluorophosphate.

A cyanobacterium, Synechococcus sp. PCC 7942 was transformed with a recombinant plasmid harboring poly-(hydroxybutyrate) (PHB)-synthesizing genes from Alcaligenes eutrophus. The acquired transformant accumulated about 1% PHB of dry cell weight in nitrogen-starved conditions. The PHB content of the transformant was kept stable during a series of batch cultures.

Mutational analysis of the poly(3-hydroxybutyrate) (PHB) depolymerase A of Pseudomonas lemoignei and of the poly(3-hydroxybutyrate) depolymerase of Alcaligenes faecalis revealed that S138 ( P. lemoignei) and S139 ( A. faecalis) are essential for activity. Both serines are part of a strictly conserved pentapeptide sequence which is present in all poly(3-hydroxybutyrate) depolymerases analyzed so far (G-L-S-S(A)-G) and which resembles the lipase ☐ of lipases and other serine hydrolases (G-X-S-X-G). Mutation of another conserved serine, namely S195 ( P. lemoignei) and S196 ( A. faecalis), resulted in mutant proteinswith almost full activity and proved that S195 and S196 are not essential for activity. The results indicate the structural and functional relationship of poly(3-hydroxybutyrate) depolymerases to the family of serine hydrolases.

La depolymerisation intracellulaire du poly(3-hydroxybutyrate) (PHB) a ete etudiee chez l'Alcaligenes eutrophus. Les granules de PHB de l'A. eutrophus ont libere du D-(-)-3-hydroxybutyrate au cours d'une incubation a 37°C. Le profil de pH de l'autodigestion des granules de PHB a revele deux pics d'activite, l'un a un pH entre 6 et 7 et l'autre a un pH plus alcalin. L'autodigestion de granules naturels de PHB a ete inhibee en presence de Triton X-100. L'activite PHB depolymerase a ete detectee dans le surnageant apres centrifugation a 100 000 x g de l'extrait cellulaire, en utilisant comme substrats les granules de PHB naturels traites a la protease qui ont ainsi perdu la majeure partie de leur activite autodigestive. Compare aux cellules carencees en PHB, l'activite PHB depolymerase soluble a augmente d'environ 20 fois ou plus pendant la synthese de PHB ; de plus, il y a eu une correlation rigoureuse entre le contenu en PHB et l'activite PHB depolymerase dans les cellules de l'A. eutrophus. Une PHB depolymerase soluble a ete partiellement purifiee et fortement inhibee en presence de fluorophosphate de diisopropyle. La PHB depolymerase dans le surnageant avait un pH optimal d'environ 8 a 9. Ces resultats indiquent que deux types de PHB depolymerase peuvent etre associes avec les granules naturels et qu'une enzyme possedant un pH optimal alcalin existe dans le surnageant de l'extrait cellulaire ; la regulation de cette enzyme semble etre exercee de pair avec la synthese de PHB dans les cellules.

Graft copolymerization of methyl methacrylate (MMA) or 2-hydroxyethyl methacrylate (HEMA) onto poly(3-hydroxybutyrate) (PHB) and its copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) {P(HBHV)} was carried out by using simultaneous radiation and preirradiation techniques from a 60Co γ-rays source. Degree of grafting (X g) of MMA onto both polymers increased as the irradiation dose increased. The X g for PHB graft-polymerized by simultaneous radiation was lower than that for the copolymer of 24 mol% HV content (24 M sample). On the contrary, X g of PHB graft-polymerized after preirradiation was higher than that of the 24 M sample. The X g depended on differences in regularity in the crystalline regions or crystallinity and rate of radical decay. Crystalline regions of PHB remained almost unchanged after grafting, while crystalline regions of the 24 M sample were partially destroyed by the introduction of grafting. Glass transition temperatures of both grafted polymers increased up to 8°C. The ...

Five extracellular PHB depolymerases of bacteria isolated from various sources were purified to electrophoretic homogeneity and compared with known extracellular PHB depolymerase fromAlcaligenes faecalis T1. The molecular mass of these enzymes were all around 40–50 kDa. Nonionic detergent, diisopropylfluorophosphate and dithiothreitol inhibited the PHB depolymerase activity of all these enzymes. Trypsin abolished PHB depolymerase activity, but not theD-3-hydroxybutyric acid dimer hydrolase activity of all the enzymes. These results showed that the basic properties of these PHB depolymerases resemble those of theA. faecalis T1 enzyme. Analysis ofN-terminal amino acid sequence of the purified enzymes revealed that these enzymes includingA. faecalis T1 enzyme fall into three groups.

The microbial copolyesters incorporating 3-hydroxyalkanoate units with different chain lengths (C4–C10) were produced from various carbon substrates by Alcaligenes eutrophus or Pseudomonas oleovorans . The enzymatic degradation processes of the poly(3-hydroxyalkanoates) (PHA) films were studied at 37°C in a phosphate buffer (pH 7·4) containing the PHA depolymerase from A. faecalis T1 . The rate of enzymatic degradation was determined by monitoring the time-dependent changes in weight loss(erosion) of polyester films. The rate was strongly dependent upon the composition of the polyesters and markedly decreased with an increase in the side-chain length of the 3-hydroxyalkanoate monomeric units. The polyester chains were finally degraded into the monomers and dimers of 3-hydroxyalkanoic acids by the PHA depolymerase.

The chemical, enzymatic and microbial degradation of bacterial and synthetic poly-β-hydroxybutyrate (PHB) and its copolymers with β-hydroxyvalerate (PHB/HV) were evaluated. Alkaline hydrolysis (7·4 and 10 pH units) was carried out at 70°C. Samples were treated with purified extracellular PHB depolymerase from Alcaligenes faecalis T1. Microbial degradation was studied using a chemostat with an inoculum of activated sludge under carbon limited conditions. Samples studied were synthetic PHB, bacterial PHB and PHB/HV. Samples were in the form of dry powders, a ‘never dried’ aqueous dispersion (latex) of native granules or films made from these materials. The microbial degradation of PHB/HV latex coated paper samples was also studied. Bacterial PHA samples degraded completely after 12 h exposure to PHB depolymerase. Degradation in a microbial environment was within a matter of days. Conversely, synthetic PHB samples which degraded more readily than bacterial PHB by alkaline hydrolysis did not degrade as readily upon exposure to a microbial environment. The extent of degradation was determined quantitatively by weight loss and was followed qualitatively by scanning electron microscopy. The degradation rate depended on sample crystallinity, tacticity, morphology and composition. In general PHB/HV films and coated paper made with the ‘never dried’ latex degraded more quickly than solvent cast films. Synthetic PHB of intermediate tacticity exposed to depolymerase enzyme biodegraded more extensively than highly isotactic synthetic PHB.

Poly(3-hydroxybutyrate) {P(3HB)} and the copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) {P(3HB-co-3HV)} were irradiated with γ-rays at 25°C in air and in vacuum. Melting points (Tm) and glass-transition temperatures (Tg) were measured by differential scanning calorimetry. Number-average molecular weights (Mn) were analyzed by gel permeation chromatography. No significant differences were observed between Tm values of P(3HB) and P(3HB-co-3HV) irradiated in air and in vacuum, which decreased almost linearly with increasing irradiation dose. The Mn values of both samples decreased sharply with increasing dose, reflecting typical random chain scission. The decrease in Mn of the sample irradiated in vacuum was smaller than that irradiated in air with the same dose, implying the occurrence of crosslinking. The Tg values for both polymers irradiated in vacuum remained almost unchanged over a wide dose range, while those irradiated in air decreased as the irradiation dose increased. Both the Tm and Tg of samples irradiated in air were inversely proportional to Mn. Biodegradability was clearly promoted with decreasing molecular weight.

The synthetic analogue of a bacterially produced polyester, poly(β-hydroxybutyrate) (PHB) was synthesized from racemic β-butyrolactone using anin situ trimethyl aluminum-water catalyst. The polymer was fractionated into samples differing in molecular weight and isotactic diad content. The latter was closely related to degree of crystallinity. The biodegradation of these fractions were examined by monitoring mass loss over time in the presence of anAlcaligenes faecalis T1 extracellular bacterial poly(β-hydroxybutyrate) depolymerase. The fraction with high isotactic diad tacticity content showed little or no degradation over a 50 hour incubation period, whereas the fraction of intermediate isotactic diad content degraded in a continuous steady fashion at a rate that was less than that for bacterial PHB. The low isotactic diad fraction underwent a rapid initial degradation, followed by no further mass loss. The presence of stereoblocks in the polymer structure of the various fractions was an influence on the degree of susceptibility towards degradation and is related to sample crystallinity.

The amino acid sequence of a peptide containing an active serine was examined with poly(3-hydroxybutyrate) (PHB) depolymerase ofAlcaligenes faecalis T1. The sequence Cys-Asn-Ala-Trp-Ala-Gly-Ser-Asn-Ala-Gly-Lys was obtained. This amino acid sequence around the active serine does not fit any reported sequence of other esterases and proteases. On the other hand, a segment of the amino acid sequence of PHB depolymerase ofA. faecalis was homologous to the type III sequence of fibronectin. Similar sequences have been reported in some type of bacterial chitinase and cellulases, and PHB depolymerase seems to have an overall similarity to these bacterial extracellular hydrolases.

The relationship between cell growth and the secretion of poly(3-hydroxybutyrate) (PHB) depolymerase by Alcaligenes faecalis T1, a gram-negative bacterium, was studied with various carbohydrates added to the medium as sole carbon sources. A. faecalis T1 could grow on many kinds of carbon sources, the growth rate depending on the carbon source species. However, among the various carbon sources tested, glucose, PHB and its metabolites, D(-)-3-hydroxybutyrate and acetoacetate, but not succinate or other carboxylic acids, caused the secretion of PHB depolymerase. In the medium containing glucose as a sole carbon source, A. faecalis T1 started to grow and to secrete PHB depolymerase after 60h of cultivation. In contrast, the growth of cells in the medium containing succinate as a sole carbon source staeted within 2h and reached a plateau level after 6h of cultivation, but the cells did not secrete the enzyme into the culture medium. However, succinate-grown cells contained a considerable amount of PHB depolymerase, the level being the same as that in glucose-grown cells. Most of the cellular PHB depolymerase was found to be localized in the membrane fractions prepared from the glucose- and succinate-grown cells. In contrast to the glucose-grown cells, the succinate-grown cells exhibited no ability to incorporate [14C]glucose, although the cells exhibited several glycolytic enzyme activities for glucose oxidation. Therefore, it seems that the glucose availability for this bacterium is dependent on the induction of some protein(s) essential for glucose uptake and that glucose metabolites such as ketone bodies are essential for PHB depolymerase secretion by A. faecalis T1.

Background: Clinicopathological studies have revealed some distinct anatomical variations in the clinical presentation of oral leukoplakia (OL). Screening studies have shown that gingival leukoplakia (GL) to be more common among the Japanese. Objective: A hospital-based case-control study was undertaken among patients diagnosed with OL to compare clinico-pathological data between GL and OL affecting other sites (n-GL). Methods: One hundred fifty-one subjects attending four city hospitals in Japan diagnosed with OL were recruited to this study. OL was diagnosed by World Health Organization criteria. They were interviewed for risk factors, clinical presentations were charted and investigated by patch testing for allergy to dental materials and by biopsy for dysplasia. Results: Eighty-eight with GL and 63 with n-GL were included in the study. GL lesions were predominantly white (93%) though n-GL leukoplakia had red (speckled) foci in 26%. Eighty percent of GL were seen in association with prosthesis or restorations. There were no significant differences by age or in tobacco and alcohol use in the two groups. Metal allergy test was positive in 58% of the GL but was not significantly higher compared to the n-GL (41%). Histopathological features showed that inflammatory cell infiltrate was predominant in n-GL group ( p = 0.021). Higher dysplasia grades were also found in n-GL ( p = 0.018). During follow-up, nine oral cancers (14%) were detected in n-GL group and among GL cases four developed oral carcinomas (5%) ( p = 0.043). Cox hazard regression analysis revealed that after adjusting for age and sex, GL group was found to have a lower malignant risk, with a hazard ratio of 0.89 (95% CI: 0.51–1.04) when compared to n-GL. Conclusions: GL was more benign compared to n-GL in Japanese subjects, though risk factors appear similar. Further study is warranted to ascertain whether trauma may play an important role for hyperkeratinization of gingival tissues and possible evolution of GL to proliferative verrucous leukoplakia in later years.

The processes of radiation vulcanization and production of protective rubber gloves for radioactive contamination are described. A newly developed sensitizing system consisting of 5 phr 2-ethylhexyl acrylate and 1 phr carbon tetrachloride was used to vulcanize natural rubber latex at 12 kGy. Transparent and soft gloves were obtained from the radiation vulcanized latex by a coagulant dipping process. The mechanical properties of the gloves meet Japanese Industrial Standard specification for protective gloves. Combustion analysis of the gloves revealed that the amount of evolved sulfur dioxide and remaining ashes are less than those from commercially available rubber gloves. A trial usage of the gloves at a nuclear power plant showed that the gloves were easy to use for delicate work without undergoing fatigue.

The rate of proliferation of porcine aortic endothelial cells (PAEC) in response to fibroblast growth factor (FGF) was largely retarded when incubated in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with either 1% fetal bovine serum (FBS) or 10% dialyzed FBS in place of 10% FBS. Proliferation of endothelial cells in low serum media in response to FGF was enhanced to the level of media containing FGF plus 10% FBS by the addition of the dialyzable fraction from bovine brain homogenates. From the bovine brain dialyzable fraction, two active components were purified and identified as hypoxanthine and inosine. Either hypoxanthine or inosine, at a dose of 5 μM in DMEM with 1% FBS, maximally increased the incorporation of [3H]thymidine into DNA of PAEC in low serum media in the presence of FGF. However, no additive effect was observed when hypoxanthine and inosine were added simultaneously. The present data indicate that the proliferative action of FGF on PAEC can be potentiated by hypoxanthine and inosine.

Tous les copolymeres etudies sont instables a l'etat fondu au dessus de 170°C, quelle que soit leur composition; leur masse moleculaire diminue rapidement avec une cinetique qui suit le modele de rupture de chaine aleatoire aux groupes esters. Les resultats suggerent que les copolyesters dont le point de fusion est inferieur a 160°C peuvent etre utilises en moulage par injection

Myoepithelioma is a rare tumor (incidence less than 1 % of all salivary gland tumors). We report a case of myoepithelioma of the hard palate.A 15-year-old man complained of a painless mass on the palate. There was a wellcircumscribed swelling, measuring 25mm in diameter, on the hard palate. It was dark blue and had an elastic soft consistency. Sagittal magnetic resonance images revealed on oval mass with iso-intensity on T 1-weighted images and medium high intensity on T 2-weighted images. The mass pressed the hard palate toward the nasal cavity, but a sheet of bone intervened between the mass and the nasal floor. The preoperative histopathological diagnosis, based on examination of an incisional biopsy specimen, was monomorphic adenoma.With the patient under general anesthesia, the tumor was removed subperiosteally with a 5-mm safety margin of mucosa. The osseous palate was hollow, but there was no bone destruction. It was therefore not resected. The tumor was 20mm in diameter and surrounded by a thin fibrous membrane.Microscopically, the tumor consisted of plasmacytoid and spindle-shaped cells with round eccentric nuclei surrounded by abundant homogenous eosinophilic cytoplasm in a fibrous stroma.Immunohistochemical studies revealed positive reactions for cytokeratin, vimentin, and S100 protein in the tumor cells.Finally, the postoperative histopathological diagnosis was myoepithelioma (combination of plasmacytoid and spindle-shaped cells).Wound healing was good, and there was no evidence of recurrence as of 3 years after the operation.

Thirteen patients with malignant lymphoma (ML) consulted our department during the 7 years from June 1990 through May 1997. The initial clinical symptoms of the patients were analyzed retrospectively.The results were as follows:1. ML accounted for 8.7% of all malignant tumors of the oral and maxillofacial region at our department.2. The subjects consisted of 12 males and 1 female. Their mean age was 62 years.3. Of the 13 patients, 11 were pathologically confirmed as having non-Hodgkin's lymphoma and 2 as having Hodgkin's disease.4. There were 3 nodal cases and 10 extranodal cases.5. The primary site of the tumor was the cervical lymph nodes in 3 cases, the gingiva in 6, the hard and soft palate in 3, and the buccal submucosa in 1.6. Only one case was clinically diagnosed as malignant lymphoma. The initial clinical diagnosis in the remaining 12 cases was other malignant tumors in 6 cases, osteomyelitis in 3, benign tumor in 2, and lymphoadenitis in 1.

Two systems, one using an (R)-(-)-3-hydroxybutyrate dehydrogenase (BDH) null mutant of Ralstonia eutropha and the other using a recombinant Escherichia coli strain containing a synthetic poly[(R)-(-)-3-hydroxybutyrate] (PHB) operon and an extracellular PHB depolymerase gene, were used for the fermentative production of (R)-(-)-3-hydroxybutyrate (3HB). The concentration of 3HB in the culture supernatant of the mutant R. eutropha system reached about 30 mM after 5 d under anaerobic conditions, although it was about 4-10 mM under aerobic conditions. On the other hand, the 3HB concentration in the culture supernatant of the recombinant E. coli system reached about 70 mM after 4 d, indicating that about 70% of the glucose added was converted to 3HB.

Poly(3-hydroxybutyrate) (PHB) is synthesized from 3-hydroxybutyryl-CoA by polyhydroxyalkanoate synthase and hydrolyzed by PHB depolymerase. In this study, we focused on the reverse reaction of polyhydroxyalkanoate synthase, and propose the possibility that PHB can be degraded through a novel process, that is thiolysis of PHB with CoASH. Polyhydroxyalkanoate synthase of Ralstonia eutropha was incubated with 14 C-labeled PHB and CoASH. The reaction mixture was fractionated by HPLC and then analyzed with a scintillation counter. The analysis revealed 3-hydroxybutyryl-CoA to be a product of the reaction. When NADP+ and acetoacetyl-CoA reductase were added to the reaction mixture, an increase in absorbance at 340 nm was observed. Native PHB inclusion bodies from R. eutropha also showed thiolytic activity. This is the first indication that polyhydroxyalkanoate synthase catalyzes both the synthesis and degradation of PHB, and that native PHB inclusion bodies has thiolytic activity.

Two D -(−)-3-hydroxybutyrate (3HB) dehydrogenases, BDH1 and BDH2, were isolated and purified from a poly(3-hydroxybutyrate) (PHB)-degradable bacterium, Ralstonia pickettii T1. BDH1 activity increased in R. pickettii T1 cells grown on several organic acids as a carbon source but not on 3HB, whereas BDH2 activity markedly increased in the same cells grown on 3HB or PHB. To examine their biochemical properties, bdh1 and bdh2 were cloned and overexpressed in Escherichia coli , and their purified products were characterized. The kinetic parameters indicate that BDH1 is more suitable for converting acetoacetate to 3HB than BDH2, whereas BDH2 is more efficient for the reverse reaction than BDH1. Thus, R. pickettii T1 contains two BDHs with different biochemical properties and physiological roles: BDH1 for cell growth on organic acids other than 3HB and BDH2 for cell growth on 3HB or PHB.

A novel intracellular poly(3-hydroxybutyrate) (PHB) depolymerase (PhaZd) of Wautersia eutropha (formerly Ralstonia eutropha ) H16 which shows similarity with the catalytic domain of the extracellular PHB depolymerase in Ralstonia pickettii T1 was identified. The positions of the catalytic triad (Ser 190 -Asp 266 -His 330 ) and oxyanion hole (His 108 ) in the amino acid sequence of PhaZd deduced from the nucleotide sequence roughly accorded with those of the extracellular PHB depolymerase of R. pickettii T1, but a signal peptide, a linker domain, and a substrate binding domain were missing. The PhaZd gene was cloned and the gene product was purified from Escherichia coli . The specific activity of PhaZd toward artificial amorphous PHB granules was significantly greater than that of other known intracellular PHB depolymerase or 3-hydroxybutyrate (3HB) oligomer hydrolases of W. eutropha H16. The enzyme degraded artificial amorphous PHB granules and mainly released various 3-hydroxybutyrate oligomers. PhaZd distributed nearly equally between PHB inclusion bodies and the cytosolic fraction. The amount of PHB was greater in phaZd deletion mutant cells than the wild-type cells under various culture conditions. These results indicate that PhaZd is a novel intracellular PHB depolymerase which participates in the mobilization of PHB in W. eutropha H16 along with other PHB depolymerases.

Polyhydroxybutyrate is a microbial polyester that can be produced from renewable resources, and is degraded by the enzyme polyhydroxybutyrate depolymerase. The crystal structures of polyhydroxybutyrate depolymerase from Penicillium funiculosum and its S39 A mutant complexed with the methyl ester of a trimer substrate of (R)-3-hydroxybutyrate have been determined at resolutions of 1.71 A and 1.66 A, respectively. The enzyme is comprised of a single domain, which represents a circularly permuted variant of the alpha/beta hydrolase fold. The catalytic residues Ser39, Asp121, and His155 are located at topologically conserved positions. The main chain amide groups of Ser40 and Cys250 form an oxyanion hole. A crevice is formed on the surface of the enzyme, to which a single polymer chain can be bound by predominantly hydrophobic interactions with several hydrophobic residues. The structure of the S39A mutant-trimeric substrate complex reveals that Trp307 is responsible for the recognition of the ester group adjacent to the scissile group. It is also revealed that the substrate-binding site includes at least three, and possibly four, subsites for binding monomer units of polyester substrates. Thirteen hydrophobic residues, which are exposed to solvent, are aligned around the mouth of the crevice, forming a putative adsorption site for the polymer surface. These residues may contribute to the sufficient binding affinity of the enzyme for PHB granules without a distinct substrate-binding domain.

Wautersia eutropha H16 (formerly Ralstonia eutropha ) mobilizes intracellularly accumulated poly(3-hydroxybutyrate) (PHB) with intracellular poly(3-hydroxybutyrate) depolymerases. In this study, a novel intracellular 3-hydroxybutyrate-oligomer hydrolase (PhaZc) gene was cloned and overexpressed in Escherichia coli . Then PhaZc was purified and characterized. Immunoblot analysis with polyclonal antiserum against PhaZc revealed that most PhaZc is present in the cytosolic fraction and a small amount is present in the poly(3-hydroxybutyrate) inclusion bodies of W. eutropha . PhaZc degraded various 3-hydroxybutyrate oligomers at a high specific activity and artificial amorphous poly(3-hydroxybutyrate) at a lower specific activity. Native PHB granules and semicrystalline PHB were not degraded by PhaZc. A PhaZ deletion mutation enhanced the deposition of PHB in the logarithmic phase in nutrient-rich medium. PhaZc differs from the hydrolases of W. eutropha previously reported and is a novel type of intracellular 3-hydroxybutyrate-oligomer hydrolase, and it participates in the mobilization of PHB along with other hydrolases.