Detection efficiency calibration for an array of fourteen HPGe detectors.

The CUP array of germanium (CAGe) is an array of fourteen high-purity germanium (HPGe) detectors. The detection efficiency of full-energy-peak emitted from the various samples assayed on the CAGe was calculated using the Monte Carlo simulation toolkit GEANT4. If the dead layer on the surface of the crystal is treated in the simulation as a continuous part of the active crystal, then the detection efficiency will be overestimated. Thus, the detection efficiency of the CAGe was adjusted using multi-nuclide source data and Monte Carlo simulations. The gamma spectra of the known activity source were obtained for each HPGe detector of the CAGe. The detection efficiency measured by the multi-source data was smaller than that of simulation data if the simulation treated the whole volume of germanium crystals as active for gamma detection. By optimizing the dead layers' thicknesses in the simulation, the detection efficiency calculated by the simulation could be matched to that of multi-source data.

Prediction of quality traits and grades of intact chicken breast fillets by hyperspectral imaging.

1. In this study, hyperspectral imaging was evaluated for its usefulness to predict quality traits and grading of intact chicken breast fillets. 2. Lightness of colour (L*) and pH of the fillets were measured as quality traits, and samples were then selected and graded to three different quality categories, i.e., dark, firm and dry (DFD), normal (NORM), and pale, soft and exudative (PSE) based on these two quality traits. Based on the prediction performance of full wavelength partial least square regression (PLSR) models, the spectral range of visible and near-infrared (Vis-NIR) was more suitable for the evaluation of quality traits and grading than the range of near-infrared (NIR). Key wavelengths of each quality trait and grade value were selected by the regression coefficient (RC) method. 3. The new key wavelength PLSR models showed good predictive performances (Rp = 0.85 and RMSEp = 2.18 for L*, Rp = 0.84, and RMSEp = 0.13 for pH, and Rp = 0.80 and RMSEp = 0.44 for quality grading). The classification accuracy for grades was 85.71% (calibration set) and 81.82% (prediction set), respectively. Finally, distribution maps showed that quality traits and grades of samples were able to be visualised. 4. These results suggested that hyperspectral imaging has the potential for quality prediction of fresh chicken meat.

Association Study of 60 Candidate Genes with Antipsychotic-induced Weight Gain in Schizophrenia Patients.

INTRODUCTION: This study aimed to investigate the association of multiple candidate genes with weight gain and appetite change during antipsychotic treatment. METHODS: A total of 233 single nucleotide polymorphisms (SNPs) within 60 candidate genes were genotyped. BMI changes for up to 8 weeks in 84 schizophrenia patients receiving antipsychotic medication were analyzed using a linear mixed model. In addition, we assessed appetite change during antipsychotic treatment in a different group of 46 schizophrenia patients using the Drug-Related Eating Behavior Questionnaire. RESULTS: No SNP showed a statistically significant association with BMI or appetite change after correction for multiple testing. We observed trends of association (P<0.05) between 19 SNPs of 11 genes and weight gain, and between 7 SNPs of 5 genes and appetite change. In particular, rs696217 in GHRL showed suggestive evidence of association with not only weight gain (P=0.001) but also appetite change (P=0.042). Patients carrying the GG genotype of rs696217 exhibited higher increase in both BMI and appetite compared to patients carrying the GT/TT genotype. DISCUSSION: Our findings suggested the involvement of a GHRL polymorphism in weight gain, which was specifically mediated by appetite change, during antipsychotic treatment in schizophrenia patients.

HMGB1 in the pathogenesis of ultraviolet-induced ocular surface inflammation.

High-mobility group box 1 (HMGB1) functions as a transcription-enhancing nuclear protein as well as a crucial cytokine that regulates inflammation. This study demonstrated that secretion of HMGB1 due to ultraviolet (UV) radiation inducing ocular surface inflammation-mediated reactive oxygen species (ROS) production. After treating conjunctival epithelial cells with UV radiation, HMGB1 was translocated from the nucleus to the cytoplasm and then eventually to the extracellular space. HMGB1 played a crucial role in UV-induced conjunctival neutrophil infiltration, which subsided when mice were pretreated with the HMGB1 inhibitors soluble receptor for advanced glycation endproducts (sRAGEs) and HMGB1 A box protein. In case of using ROS quencher, there was decrease in UV-induced HMGB1 secretion in conjunctival epithelial cells and mice. Considering that UV-induced chronic inflammation causes ocular surface change as pterygium, we have confirmed high HMGB1 translocation and ROS expression in human pterygium. Our findings therefore revealed a previously unknown mechanism of UV-induced ocular inflammation related to ROS and HMGB1 suggesting a new medical therapeutic target.

Dynamic and static shell properties of white and brown shell eggs exposed to modified pressure microcrack detection technology.

Dynamic and static shell properties of eggs provide important insight to egg quality. Understanding how processing and handling procedures affect both dynamic and static shell properties can enhance the safety and quality of eggs reaching the consumer. A study was conducted to determine if dynamic shell properties were altered due to modified pressure microcrack detection technology exposure in brown and white shell eggs. Three replicates of 100 eggs each of brown and white retail shell eggs were conducted. Dynamic stiffness (K(dyn)) and egg weight were monitored immediately before and after microcrack detection. No changes in K(dyn) or egg weight were detected for either shell color. Static compression shell strength and deformation were subsequently monitored and a correlation analysis conducted. A strong correlation (R(2) = 0.53; P < 0.0001) between K(dyn) and static compression shell strength was seen for extra large white shell eggs. A smaller (R(2) = 0.31; P < 0.0001) correlation was found for large brown eggs. The use of modified pressure microcrack detection technology did not affect shell dynamic properties.

Salmonella contamination in shell eggs exposed to modified-pressure imaging for microcrack detection.

Microcracks in egg shells are a food safety risk and are difficult for professional human graders to detect. Modified-pressure imaging technology with 99.6% accuracy has been developed to detect microcracks. This study was conducted to determine whether the microcrack detection system would increase penetration of Salmonella into egg contents or lead to cross-contamination within the system. Thirty dozen grade A large white retail eggs were used for each of 3 replicates. Cracked eggs were removed and 72 eggs/replicate were dip inoculated in buffered peptone water containing 10(5) cfu/mL of nalidixic acid-resistant Salmonella Typhimurium (ST), whereas 144 eggs were dipped in sterile buffered peptone water. All eggs were incubated overnight at 25°C before imaging. Forty-five eggs of each treatment were imaged in the following order: control, inoculated, control. Imaged and nonimaged eggs from each treatment were used for cultural analysis of a shell rinse, shell emulsion, and contents sample for each egg. The ST levels were monitored on brilliant green sulfa agar with 200 mg/L of nalidixic acid. Egg contents were also enriched to determine the prevalence of ST in low levels. Salmonella Typhimurium was not detected on or in any of the control eggs, including the eggs imaged after the inoculated eggs. The highest level of ST was detected in inoculated shell emulsions (4.79 log cfu/mL). No differences in ST levels were found for any sample location between imaged and nonimaged inoculated eggs. Therefore, the modified-pressure imaging system for microcrack detection did not result in microbial cross-contamination or increase the level of microbial penetration in inoculated eggs. The imaging system can be used to assess eggs for cracks without negative food safety implications.

Supine linac treatment versus tomotherapy in craniospinal irradiation: planning comparison and dosimetric evaluation.

Craniospinal irradiation (CSI) is the standard treatment of primary intracranial tumour with risk of leptomeningeal dissemination. However, supine setup field-in-field technique does not need inter-fractional junction shift. Recently, the studies of CSI with tomotherapy showed excellent target coverage and tolerable normal organ dose in paediatric patients. The planning comparison and dosimetric difference between conventional radiotherapy and tomotherapy are presented. Three patients with central nervous system germinoma received supine CSI treatment. Normal tissue complication probability calculation was performed for parotid gland, kidney, lens, small bowel, ovary and testis. Homogenous vertebral body coverage for tomotherapy compared with conformal radiotherapy was found. The mean dose to each parotid gland decreased by 7.3 and 10 Gy, respectively, with tomotherapy. The volume of oesophagus and small bowel receiving >10 Gy was significantly lower. The V2, V5, V10 and V20 of the lungs are 81.6, 12.4, 2.3 and 0 % with tomotherapy. Tomotherapy showed excellent homogenous dose distribution through the craniospinal axis (PTV) and higher conformity index.

Modified pressure imaging for egg crack detection and resulting egg quality.

Cracks in the shell surface compromise the primary barrier for external microbial contamination of the egg. Microcracks are very small cracks in the shell surface that are difficult to detect by human graders. New technology has been developed that uses modified pressure and imaging to detect microcracks in eggs. Research has shown the system to have an accuracy of 99.6% in detecting both cracked and intact eggs. A study was undertaken to determine if quality differences existed between modified pressure imaged and control eggs during extended cold storage. Three replicates were conducted with eggs stored at 4 degrees C for 5 wk with weekly quality testing. The physical quality factors monitored were Haugh units, albumen height, egg weight, shell strength, vitelline membrane strength and elasticity, and whole egg total solids. All measurements were conducted on individual eggs (12/treatments per replicate) each week with the exception of whole egg solids, which were determined from 3 pools (4 eggs each)/treatment per replicate each week. Percentage of whole egg total solids was the only significant difference (P < 0.05) between treatments (23.65% modified pressure imaged and 23.47% control). There was a significant difference (P < 0.05) for egg weight between replicates (60.82, 58.02, and 60.58 g for replicates 1, 2, and 3, respectively). Therefore, imaging eggs in the modified pressure system for microcrack detection did not alter egg quality during extended cold storage. Utilizing the modified pressure crack detection technology would result in fewer cracked eggs reaching the consumer, consequently enhancing food safety without affecting product quality.

Application of a glass rod detector for the output factor measurement in the CyberKnife.

A radiophotoluminescent glass rod detector has recently become commercially available. We evaluated the feasibility of the commercial glass rod as a new detector for measuring output factors in the CyberKnife. The glass rod detector was irradiated in a water phantom using a holder stand, which was specially designed for this study. The holder was composed of a PMMA tube with an attached vertical bar for the glass rod detector. The measured output factors obtained with the glass rod detector were compared with measurements made with a pinpoint ionization chamber, a diode, and a radiochromic film. The measured relative output factors obtained with the glass rod detector agreed with other detectors within 1.0% for collimator sizes larger than 20mm. However, it was observed that the differences between the output factors measured with the glass rod detector and those obtained with the pinpoint chamber increased rapidly as the collimator size decreased. The relative output factors measured with the diode were consistently higher than those obtained using other detectors for the collimators sizes less than 10mm in diameter. The glass rod detector results were in good agreement with those obtained from the radiochromic EBT film over the entire range of collimator sizes.

No association of the TGF-beta1 gene polymorphisms with the renal progression in autosomal dominant polycystic kidney disease (ADPKD) patients.

BACKGROUND: Two genetic loci, PKD I and PKD2, have been identified as being responsible for ADPKD, and PKD1 is known to be associated with a poor prognosis. However, the presence of an intrafamilial study clinical diversity suggests that there are disease-modifying loci. Because the mechanism ofthe renal failure in ADPKD includes a cystic growth and tubulointerstitial atrophy and fibrosis, we studied the associations between 2 polymorphisms in the TGF-beta1 gene, which are known to be associated with chronic tubulointerstitial inflammation, and ADPKD progression in Korean patients. PATIENTS AND METHODS: One hundred and twenty-five individuals who had ADPKD and 47 normal control subjects were genotyped by PCR-RFLP, the T869C (Leu10Pro) variant of TGF-beta gene leader sequence was discriminated with MspA1I and the G915C (Arg25Pro) variants with Bg1I. Statistical significances were determined using the Chi-square test. RESULTS: The distribution of the alleles for the TGF beta1 Leu10Pro polymorphism in ADPKD was: T 54%, C 46%, which was similar to the Korean (56: 44, p = 0.887) and Western controls (65: 35). In addition, no differences were found between the ESRD and the non-ESRD groups (p = 0.888) or the early hypertension and the normotension groups (p = 0.249). The distribution of alleles for the TGF beta1 Arg25Pro polymorphism showed only the GG type which was different from the Western population controls (G:C = 90:10, p = 0.000). CONCLUSIONS: Our results suggest that the polymorphism at Arg25Pro of TGF-beta1 in the Korean population has an allele distribution different from that ofthe Western population and that the polymorphism at Leu10Pro of TGF-beta1 has no association with the renal progression in Korean ADPKD patients.

Biosynthesis and local sequence specific degradation of poly(3-hydroxyvalerate-co-4-hydroxybutyrate) in Hydrogenophaga pseudoflava.

A novel copolymer that consisted of 3-hydroxyvalerate and 4-hydroxybutyrate, P(3HV-co-4HB), was synthesized in Hydrogenophaga pseudoflava by growing it in media containing gamma-valerolactone and gamma-butyrolactone as a carbon source. The monomer ratio in the copolymer was changed by altering the feed ratio of the two lactones. The cultivation technique was composed of three steps: the first-step for high cell production in Luria-Bertani medium, the second-step for intracellular degrading removal of poly(3-hydroxybutyrate) (P(3HB)), which was formed in the first step, by culturing the cells in carbon-source-free medium, and the final step for accumulation of P(3HV-co-4HB) in a mixed lactone medium. All the P(3HV-co-4HB) copolymers contained less than 1 mol % of 3HB unit. These copolymers were characterized by NMR spectroscopy, differential scanning calorimetry, wide-angle X-ray diffraction, and first-order kinetic analysis of intracellular degradation. The copolymer with an approximately equal ratio of the comonomers was found amorphous. The NMR microstructural analysis showed that the copolymers contained appreciable amounts of 3HV-rich or 4HB-rich chains. The (13)C NMR splitting patterns associated with the four carbons in the 4HB unit of P(3HV-co-4HB) bear close resemblance to those observed in the 4HB unit of P(3HB-co-4HB). The signals arising from the carbons in the 3HV unit of P(3HV-co-4HB) split in a manner similar to those in the 3HB unit of P(3HB-co-4HB). Thus the sequences were assigned by comparing the NMR splittings for P(3HV-co-4HB) with those for P(3HB-co-4HB) and P(3HB-co-3HV). The sequence assignment was further checked by comparing the signal intensities before and after degradation of the copolymers. This was considered reasonable because the H. pseudoflava intracellular PHA depolymerase is more specific to the 3HV unit than to the 4HB unit, which was also confirmed by the higher degradation rate constant for the 3HV unit in the first-order kinetic analysis.

Intracellular degradation of two structurally different polyhydroxyalkanoic acids accumulated in Pseudomonas putida and Pseudomonas citronellolis from mixtures of octanoic acid and 5-phenylvaleric acid.

From a set of mixed carbon sources, 5-phenylvaleric acid (PV) and octanoic acid (OA), polyhydroxyalkanoic acid (PHA) was separately accumulated in the two pseudomonads Pseudomonas putida BM01 and Pseudomonas citronellolis (ATCC 13674) to investigate any structural difference between the two PHA accumulated under a similar culture condition using one-step culture technique. The resulting polymers were isolated by chloroform solvent extraction and characterized by fractional precipitation and differential scanning calorimetry. The solvent fractionation analysis showed that the PHA synthesized by P. putida was separated into two fractions, 3-hydroxy-5-phenylvalerate (3HPV))-rich PHA fraction in the precipitate phase and 3-hydroxyoctanoate (3HO)-rich PHA fraction in the solution phase whereas the PHA produced by P. citronellolis exhibited a rather little compositional separation into the two phases. According to the thermal analysis, the P. putida PHA exhibited two glass transitions indicative of the PHA not being homogeneous whereas the P. citronellolis PHA exhibited only one glass transition. It was found that the structural heterogeneity of the P. putida PHA was caused by a significant difference in the assimilation rate between PV and OA. The structural heterogeneity present in the P. putida PHA was also confirmed by a first order degradation kinetics analysis of the PHA in the cells. The two different first-order degradation rate constants (k(1)), 0.087 and 0.015/h for 3HO- and 3HPV-unit, respectively, were observed in a polymer system over the first 20 h of degradation. In the later degradation period, the disappearance rate of 3HO-unit was calculated to be 0.020 h. The k(1) value of 0.083/h, almost the same as for the 3HO-unit in the P. putida PHA, was obtained for the P(3HO) accumulated in P. putida BM01 grown on OA as the only carbon source. In addition, the k(1) value of 0.015/h for the 3HPV-unit in the P. putida PHA, was also close to 0.019/h for the P(3HPV) homopolymer accumulated in P. putida BM01 grown on PV plus butyric acid. On the contrary, the k(1) values for the P. citronellolis PHA were determined to be 0.035 and 0.029/h for 3HO- and 3HPV-unit, respectively, thus these two relatively close values implying a random copolymer nature of the P. citronellolis PHA. In addition, the faster degradation of P(3HO) than P(3HPV) by the intracellular P. putida PHA depolymerase indicates that the enzyme is more specific against the aliphatic PHA than the aromatic PHA.

Potential of adenoviral p53 gene therapy and irradiation for the treatment of malignant gliomas.

We investigated the combined effects of p53 gene transfer and irradiation and its still unclear interaction mechanism in human gliomas. Four human glioma cell lines expressing mutant type p53 (U373 and A172) and wild-type p53 (D54MG and EFC-2) were transfected by adenoviral vectors bearing p53 gene at 50 multiplicity of infection. Two days after transfection, cells were irradiated (3, 6, and 9 Gy). The cytotoxicity was evaluated by clonogenic assay. The quantitative analysis of apoptosis and cell cycle analysis were performed using flow cytometry. Irradiation combined with adenoviral p53 transfection significantly increased cytotoxicity, which was additive in cell lines with wild-type p53 and more than additive in cell lines with mutant p53. The combination of two modalities increased the apoptotic population by 14% in A172 cells and 20% in D54 MG cells, which were the sum of apoptosis from each modality. Adenoviral p53 transfection increased the G1 phase fraction and concomitant decrease of radioresistant S phase fraction in A172 and D54MG cells. Our study demonstrated that p53 gene transfer combined with irradiation increased absolute cytotoxicity in human glioma cells used in this experiment. The interaction mechanism for increased cytotoxicity involved, in part, increased apoptosis and change of cell cycle profile.

Hetero-Diels-Alder reactions of alpha-aryl-beta-monohalo-alpha-nitrosoethylenes: diastereoselective synthesis of 6-substituted 3-aryl-4-halo-5,6-dihydro-4H-1,2-oxazines.

Treatment of alpha-aryl-beta-bromo(or chloro)-alpha-nitrosoethylene, prepared in situ from alpha-monobromo(or chloro)ketoximes and sodium carbonate in ether at rt, with allytrimethylsilane afforded exclusively trans-(4S,6S)- and trans-(4R,6R)-3-aryl-4-halo-6-[(trimethylsilyl)methyl]-5,6-dihydro-4H-1,2-oxazines 10 albeit in low yields. Similar treatment of beta-halo-alpha-nitrosoethylenes with ethyl vinyl ether, however, gave single stereoisomers, i.e., cis-(4S,6S)- and cis-(4R,6R)-6-ethoxy-4-halo-5,6-dihydro-4H-1,2-oxazines 11, in moderate to good yields. The result is in contrast to the reported predominant formation of trans-11a by a radical reaction. On the other hand, similar reactions with tert-butyl vinyl ether at 30 degrees C gave diastereomeric mixtures of cis-(4S,6S)-, cis-(4R,6R)-, trans-(4S,6R)-, and trans-(4R,6S)-6-(tert-butoxy)-4-halo-5,6-dihydro-4H-1,2-oxazines 12. In contrast to compounds 11, the major isomers have (4S,6R) and (4R,6S) configurations. The tendency of a [4 + 2] cycloaddition reaction is consistent with that observed in the Diels-Alder reaction with inverse-electron demand. The stereochemistries of compounds 10-12 were assigned on the basis of the (1)H NMR coupling constants, which were unambiguously determined by the decoupling experiments. All reactions leading to compounds 10-12 proceed with very high regioselectivity. Diastereoselectivity and high regioselectivity are understood in terms of the frontier orbital method. It has been found that cis-12g is isomerized to a mixture of stereoisomers in favor of the trans-isomer in the presence of HClO(4) (72%) in CHCl(3) at rt.

Accumulation of polyhydroxyalkanoic acid containing large amounts of unsaturated monomers in Pseudomonas fluorescens BM07 utilizing saccharides and its inhibition by 2-bromooctanoic acid.

A psychrotrophic bacterium, Pseudomonas fluorescens BM07, which is able to accumulate polyhydroxyalkanoic acid (PHA) containing large amounts of 3-hydroxy-cis-5-dodecenoate unit up to 35 mol% in the cell from unrelated substrates such as fructose, succinate, etc., was isolated from an activated sludge in a municipal wastewater treatment plant. When it was grown on heptanoic acid (C(7)) to hexadecanoic acid (C(16)) as the sole carbon source, the monomer compositional characteristics of the synthesized PHA were similar to those observed in other fluorescent pseudomonads belonging to rRNA homology group I. However, growth on stearic acid (C(18)) led to no PHA accumulation, but instead free stearic acid was stored in the cell. The existence of the linkage between fatty acid de novo synthesis and PHA synthesis was confirmed by using inhibitors such as acrylic acid and two other compounds, 2-bromooctanoic acid and 4-pentenoic acid, which are known to inhibit beta-oxidation enzymes in animal cells. Acrylic acid completely inhibited PHA synthesis at a concentration of 4 mM in 40 mM octanoate-grown cells, but no inhibition of PHA synthesis occurred in 70 mM fructose-grown cells in the presence of 1 to 5 mM acrylic acid. 2-Bromooctanoic acid and 4-pentenoic acid were found to much inhibit PHA synthesis much more strongly in fructose-grown cells than in octanoate-grown cells over concentrations ranging from 1 to 5 mM. However, 2-bromooctanoic acid and 4-pentenoic acid did not inhibit cell growth at all in the fructose media. Especially, with the cells grown on fructose, 2-bromooctanoic acid exhibited a steep rise in the percent PHA synthesis inhibition over a small range of concentrations below 100 microM, a finding indicative of a very specific inhibition, whereas 4-pentenoic acid showed a broad, featureless concentration dependence, suggesting a rather nonspecific inhibition. The apparent inhibition constant K(i) (the concentration for 50% inhibition of PHA synthesis) for 2-bromooctanoic acid was determined to be 60 microM, assuming a single-site binding of the inhibitor at a specific inhibition site. Thus, it seems likely that a coenzyme A thioester derivative of 2-bromooctanoic acid specifically inhibits an enzyme linking the two pathways, fatty acid de novo synthesis and PHA synthesis. We suggest that 2-bromooctanoic acid can substitute for the far more expensive (2,000 times) and cell-growth-inhibiting PHA synthesis inhibitor, cerulenin.

Non-linear dynamic analysis of clozapine-induced electroencephalographic changes in schizophrenic patients--a preliminary study.

1. In order to find the electroencephalographic (EEG) parameters that reflect the effect of clozapine in schizophrenic patients, the authors applied various non-linear analyses on multi-channel EEG data drawn from patients before and after a therapeutic trial of clozapine. 2. The correlation dimension was difficult to extract from our limited time series EEG data and the authors did not find a meaningful association with clozapine use. The primary Lyapunov exponent could be reliably calculated but also did not reflect the effect of clozapine. 3. However, the mutual cross-prediction (MCP) algorithm showed potentially meaningful results. The driving system was shifted to the frontal channels after a 4-week trial with clozapine. Moreover, MCP might have a value as a predictor of treatment response. 4. Although preliminary in nature, the MCP might have greater power for interpreting complex changes from channel to channel in EEG induced by clozapine.

Preparation and characterization of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) microspheres for the sustained release of 5-fluorouracil.

Biodegradable microspheres were prepared with poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV, 85:15 by mole ratio of hydroxybutyrate to hydroxyvalerate) by an water-in-oil-in-water (W/O/W), oil-in-water (O/W) and oil-in-oil (O/O) solvent evaporation method for the sustained release of anti-cancer drug, 5-fluorouracil (5-FU) with controlling the fabrication conditions. The shape of microspheres prepared was relatively rough due to highly crystalline property of PHBV and spherical. The efficiency of 5-FU loading into the PHBV microsphere with O/O method was over 80% compared to that 7% for microspheres by O/W method and below 1% for microspheres by a conventional W/O/W method. However, the most desirable release pattern can be achieved from the O/W method due to the cosolvent effect. The effects of preparation conditions such as the type and amount of surfactant, initial amount of loaded drug, the temperature of solvent evaporation, and etc. on the morphology for W/O/W method were investigated. Possible mechanisms of the desirable sustained release pattern for O/W system have been proposed.

Surface modification of polyhydroxyalkanoate films and their interaction with human fibroblasts.

A poly(3-hydroxybutylate-co-hydroxyvalerate) (PHA) film containing 34 mol.% 3-hydroxyvalerate (Biopol D600P) was prepared by the solvent cast method using a 10 wt.% chloroform solution of PHA. The PHA film was exposed to an oxygen plasma glow discharge to produce peroxides on its surfaces. These peroxides were then used as catalysts for the polymerization of acrylic acid (AA) in order to prepare carboxyl group-introduced PHA (PHA-C). Insulin-immobilized PHA was prepared using the coupling reaction of PU-C with insulin. The surface-modified PHAs were then characterized by attenuated total reflection Fourier transform infrared spectroscopy, electron spectroscopy for chemical analysis, and a contact angle goniometer. The amounts of insulin directly coupled to the carboxyl groups on PHA-C and coupled to the terminus amino groups of the grafted polyethylene oxide were 2.9 and 0.8 microg cm(-2), respectively. The PHA water contact angle (75 degrees ) decreased with AA grafting (33 degrees ) and insulin immobilization (31 degrees ), thereby exhibiting the increased hydrophilicity of the modified PHAs. When compared with PHA and PHA-C, the proliferation of human fibroblasts in the presence of serum was significantly accelerated on the insulin-immobilized PHAs.

Surface characterization and in vitro blood compatibility of poly(ethylene terephthalate) immobilized with insulin and/or heparin using plasma glow discharge.

Poly(ethylene terephthalate)(PET) film was exposed to oxygen plasma glow discharge to produce peroxides on its surfaces. These peroxides were then used as catalysts for the polymerization of acrylic acid (AA) in order to prepare a carboxylic acid group-introduced PET (PET-AA). Insulin and heparin co-immobilized PET (PET-I-H) was prepared by the grafting of poly(ethylene oxide) (PEO) on to PET-AA, followed by reaction first with insulin and then heparin. These surface-modified PETs were characterized by attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy, electron spectroscopy for chemical analysis (ESCA), and a contact angle goniometer. The concentration of the heparin (1.23 microg/cm2) bound to the PEO-grafted PET (PET-PEO) was higher than that (0.77 microg/cm2) on the insulin-immobilized PET (PET-In). The blood compatibilities of the surface-modified PETs were examined using in vitro thrombus formation, plasma recalcification time (PRT), activated partial thromboplastin time (APTT), and platelet adhesion and activation. In the experiment with plasma proteins, the PRT and APTT were significantly prolonged for both the heparin-immobilized PET (PET-He) and the PET-I-H, suggesting the binding of immobilized heparin to antithrombin III. The percentage of platelet adhesion slightly increased with the introduction of AA on the PET surfaces, decreased with the introduction of PEO and insulin, and decreased further with the immobilization of heparin. The release of serotonin was highly suppressed on PET-He and PET-I-H, and on surface-modified PETs the percentage of its release increased with an increase in platelet adhesion.

Local sequence dependence of polyhydroxyalkanoic acid degradation in Hydrogenophaga pseudoflava.

The first order intracellular degradation of various polyhydroxyalkanoic acid (PHA) inclusions in Hydrogenophaga pseudoflava cells was investigated by analyzing the compositional and microstructural changes of the PHA using gas chromatography, (13)C NMR spectroscopy, and differential scanning calorimetry. Two types of PHA, copolymers and blend-type polymers, were separately accumulated in cells for comparison. The constituent monomers were 3-hydroxybutyric acid (3HB), 4-hydroxybutyric acid (4HB), and 3-hydroxyvaleric acid (3HV). It was found that the 3HB-4HB copolymer was degraded only when the polymer contained a minimal level of 3HB units. With the cells containing a 3HB/4HB blend-type polymer, only poly(3HB) was degraded, whereas poly(4HB) was not degraded, indicating the totally inactive nature of the intracellular depolymerase against poly(4HB). On the basis of the magnitude of the first order degradation rate constants, the relative substrate specificity of the depolymerase toward the constituting monomer units was determined to decrease in the order 3HB > 3HV > 4HB. (13)C NMR resonances of the tetrad, triad, and dyad sequences were analyzed for the samples isolated before and after degradation experiments. The results showed that the intracellular degradation depended on the local monomer sequence of the copolymers. The relative substrate specificity of the depolymerase determined from the NMR local sequence analysis agreed well with that obtained from the kinetics analysis. It is suggested that, without isolation and purification of the intracellular PHA depolymerase and "native" PHA substrates, the relative specificity of the enzyme as well as the microstructural heterogeneity of the PHA could be determined by measuring in situ the first order degradation rate constants of the PHA in cells.