TEM study of the morphology of Mn2+ -doped calcium hydroxyapatite and beta-tricalcium phosphate

Mn-doped carbonated hydroxyapatites (HA) were prepared by precipitation method. Ca-deficient HA samples were obtained by this method with the characteristic hexagonal apatite structure. Scanning transmission electron microscopy (STEM) of two HA samples with two different Mn content has shown that their morphology depends on their Mn content. In case of relatively low (0.73%) Mn content (HAMn1), platelet crystals about micron size and needle-like crystals up to 100 nm were observed, while with 1.23% Mn (HAMn2) crystals were smaller, needle-like and with sizes up to 400 nm only. Mn-doped TCP samples were prepared by two methods. In one case it was obtained by direct solid-state reaction with the characteristic rhombohedral structure of beta-TCP and with composition of Ca(2.7)Mn(0.3)(PO(4))(2). TEM pictures of crystals of this sample were tens of micron and submicron size with visible faces. Crystals of beta-TCP obtained by high temperature partial transformation of sample HAMn2 to beta-TCP were found by TEM to be smaller, micron sized, drop-like shaped, sensitive to beam radiation. These results indicate that the morphology of Mn doped beta-tricalcium phosphate samples depends on the method of their preparation. Morphological properties of HA and TCP are discussed and it is suggested that the smaller and less perfect HA crystals with the higher Mn-content as well as the less perfect TCP crystals obtained by transformation of HA to TCP might be of more biocompatible character.     

Chain scission of hyaluronan by carbonate and dichloride radical anions: Potential reactive oxidative species in inflammation?

The reactions of the carbonate and dichloride radical anions, CO3- and Cl2-, with the extracellular matrix glycosaminoglycan hyaluronan (HA) have been studied using the kinetic technique of pulse radiolysis and also by steady-state irradiation combined with gel permeation chromatography/multiangle laser light scattering(gpc/MALLS) to measure the rates of reaction with HA and the yield of HA chain scission, respectively. For comparison, the same measurements were made for the reactions of the free radicals *OH, Br2*-, and N3*. The carbonate and dichloride radical anions were found to react relatively quickly with HA (7.0 x 10(5) and 6.9 x 10(6) dm3 mol(-1) s(-1), respectively) although they are much less reactive than the hydroxyl radical, *OH. Significant yields (20 and 38%, respectively) of chain scission of HA by these radical anions were also determined from the gpc/MALLS experiments, providing some support for their potential participation in the depolymerization of HA in vivo. These results are compared with data obtained for the other free radicals (hydroxyl, azide radicals, and dibromide radical anions) investigated in this study in order to gain an insight into their mechanism of reaction with HA. Earlier chain scission yields of HA by hydroxyl radicals determined by the authors have also been revised using the gpc/MALLS technique employed in the current study. The yields of 52% (absence of air) and 44% (in air) are much lower than the previous values. In the current study, the effect of oxygen on the yields of HA chain breaks is discussed in terms of the reactivity of HA peroxyl radicals in the presence of superoxide radical anions. The relevance of the results of this study to mechanisms of inflammation is discussed.     

Novel biodegradable aliphatic poly(butylene succinate-co-cyclic carbonate)s bearing functionalizable carbonate building blocks: II. Enzymatic biodegradation and in vitro biocompatibility assay

In a previous study, we have reported chemical synthesis of novel aliphatic poly(butylene succinate-co-cyclic carbonate) P(BS-co-CC)s bearing various functionalizable carbonate building blocks, and this work will continue to present our new studies on their enzymatic degradation and in vitro cell biocompatibility assay. First, enzymatic degradation of the novel P(BS-co-CC) film samples was investigated with two enzymes of lipase B Candida Antartic (Novozyme 435) and lipase Porcine Pancreas PPL, and it was revealed that copolymerizing linear poly(butylene succinate) PBS with a functionalizable carbonate building block could remarkably accelerate the enzymatic degradation of a synthesized product P(BS-co-CC), and its biodegradation behavior was found to strongly depend on the overall impacts of several important factors as the cyclic carbonate (CC) comonomer structure and molar content, molar mass, thermal characteristics, morphology, the enzyme-substrate specificity, and so forth. Further, the biodegraded residual film samples and water-soluble enzymatic degradation products were allowed to be analyzed by means of proton nuclear magnetic resonance (1H NMR), gel permeation chromatograph (GPC), differential scanning calorimeter (DSC), attenuated total reflection FTIR (ATR-FTIR), scanning electron microscope (SEM), and liquid chromatograph-mass spectrometry (LC-MS). On the experimental evidences, an exo-type mechanism of enzymatic chain hydrolysis preferentially occurring in the noncrystalline domains was suggested for the synthesized new P(BS-co-CC) film samples. With regard to their cell biocompatibilities, an assay with NIH 3T3 mouse fibroblast cell was conducted using the novel synthesized P(BS-co-CC) films as substrates with respect to the cell adhesion and proliferation, and these new biodegradable P(BS-co-CC) samples were found to exhibit as low cell toxicity as the PLLA control, particularly the two samples of poly(butylene succinate-co-18.7 mol % dimethyl trimethylene carbonate) P(BS-co-18.7 mol % DMTMC) and poly(butylene succinate-co-21.9 mol % 5-benzyloxy trimethylene carbonate) P(BS-co-21.9 mol % BTMC) were interestingly found to show much better cell biocompatibilities than the PLLA reference.     

La ions in precipitated hydroxyapatites.

Hydroxyapatites were synthesized by precipitation from an aqueous solution with La3+ (0-0.75%) and with carbonate (0-6.1%) at controlled pH 7.0. Uptake of La3+ was 90-95% complete. Relatively low Ca/P (1.54-1.63) ratios were attributed to nonstoichiometry. Carbonate in samples was identified by IR spectroscopy as B-type carbonate. Lattice parameters of the hexagonal apatite structure were not affected by the La3+ content. Noncarbonated samples heated to 800 degrees C transform partially to beta-Ca3(PO4)2. Thermogravimetric analysis showed release of 0.4 mol adsorbed and 1 mol crystalline water up to 400 degrees C and decomposition of carbonate up to 900 degrees C in the samples. Luminescence data obtained for Gd-containing hydroxyapatites prove that Gd3+ ions are not incorporated in the precipitated hydroxyapatite. These findings suggest that, in the La-containing samples, La3+ is surface absorbed and not incorporated in hydroxyapatite.     

Polyhydroxybutyrate/Hydroxyapatite Highly Porous Scaffold for Small Bone Defects Replacement in the Nonload-bearing Parts

In the present work,Polyhydroxybutyrate (PHB)/Hydroxyapatite (HA) porous composites (10％,20％,30 ％,40％,50％ weight HA) were obtained by sintering.PHB/20％ HA optimally combines satisfactory mechanical properties with a high content of the bioactive component (HA).Porous PHB/20％ HA scaffolds have shown high mechanical properties (compressive strength of 106 MPa and Young's modulus of 901 MPa).A high volume fraction of interconnected pores (＞ 50 vol.％) was achieved with pore size of 50 μm-500 μm.Biocompatibility of porous pure PHB and PHB/20％HA,as its osseointegration were assessed in vitro and after implantation in laboratory animals.PHB/20％ HA (-5％ ± 0.9％) and pure PHB (-3％ ± 1.4％) samples after 24 hours of incubation with human leucocytes showed no significant level of cytotoxicity when p =0.648 (p-value).In vitro massive adhesion of mouse Multipotent Mesenchymal Stromal Cells (MMSC) to the surface of both porous samples was shown.PHB/20％ HA induced more intensive MMSC proliferation compared to pure PHB,which are 31％ ± 6.1％ and 20％ ± 5.7 ％ respectively when p =0.039.We observed the resorption (implant surface area was reduced by 49 ％) and integration of the porous PHB/20％ HA samples into surrounding tissues after 30 days of implantation.The signs of osteoclasts accumulation,neo-angigenesis and new bone formation were observed,which make PHB/20％ HA promising for bone tissue engineering.     

Effect of sodium polyacrylate on the hydrolysis of octacalcium phosphate

Octacalcium phosphate (OCP) hydrolysis into hydroxyapatite (HA) has been investigated in aqueous solutions at different concentrations of sodium polyacrylate (NaPA). In the absence of the polyelectrolyte, OCP undergoes a complete transformation into HA in 48 h. The hydrolysis is inhibited by the polymer, which is significantly adsorbed on the crystals, up to about 22 wt.%. A polymer concentration of 10(-2) mM is sufficient to cause a partial inhibition of OCP to HA transformation, which is completely hindered at higher concentrations. The small platelet-like crystals in the TEM images of partially converted OCP can display electron diffraction patterns characteristic either of OCP single crystals or of polycrystalline HA, whereas the much bigger plate-like crystals exhibit diffraction patterns characteristic of OCP single crystals. The polyelectrolyte adsorption on OCP crystals is accompanied by an increase of their mean length and by a significant reduction of the coherence length of the perfect crystalline domains along the c-axis direction. It is suggested that the carboxylate-rich polyelectrolyte is adsorbed on the hydrated layer of the OCP (100) face, thus inhibiting its in situ hydrolysis into HA.     

Low-temperature glass transition in proteins

The viscoelastic properties of solid samples (crystals, amorphous films) of hen egg white lysozyme, bovine serum albumin, and sperm whale myoglobin were studied in the temperature range of 100–300 K at different hydration levels. Decreasing the temperature was shown to cause a steplike increase in the Young's modulus of highly hydrated protein samples (with water content exceeding 0.3 g/g dry weight of protein) in the temperature range of 237–251 K, followed by a large increase in the modulus in the broad temperature interval of 240–130 K, which we refer to as a mechanical glass transition. Soaking the samples in 50% glycerol solution completely removed the steplike transition without significantly affecting the glass transition. The apparent activation energy determined from the frequency dependence of the glass-transition temperature was found to be 18 kcal/mol for wet lysozyme crystals. Lowering the humidity causes both the change of the Young's modulus in response to the transition and the activation energy to decrease. The thermal expansion coefficient of amorphous protein films also indicates the glass transition at 150–170 K. The data presented suggest that the glass transition in hydrated samples is located in the surface layer of proteins and related to the immobilization of the protein groups and strongly bound water.     

The association of carbohydrate with tubulin and in vitro assembled microtubules from bovine brain.

Phosphocellulose-purified tubulin (PC tubulin) was analyzed for neutral and amino sugar content, which was found to be 8.3 +/- 0.11 and 0.8 +/- 0.02 mol/mol dimer, respectively. A histochemical-electron-microscopic investigation was undertaken to attempt to localize carbohydrate associated with polymerized microtubules (MT). Outer diameters of MT assembled in vitro from bovine brain MT protein (tubulin and microtubule associated proteins) were found to increase upon treatment with ruthenium red, Alcian blue, and lanthanum hydroxide, which have been reported to possess specificity for complex carbohydrates. Concanavalin A-reactive sites were detected on the surface and in the lumen of MT assembled from MT protein and from PC tubulin.     

In situ analysis of mineral content and crystallinity in bone using infrared micro-spectroscopy of the nu(4) PO(4)(3-) vibration.

Measurements of bone mineral content and composition in situ provide insight into the chemistry of bone mineral deposition. Infrared (IR) micro-spectroscopy is well suited for this purpose. To date, IR microscopic (including imaging) analyses of bone apatite have centered on the nu(1),nu(3) PO(4)(3-) contour. The nu(4) PO(4)(3-) contour (500-650 cm(-1)), which has been extensively used to monitor the crystallinity of hydroxyapatite in homogenized bone samples, falls in a frequency region below the cutoff of the mercury-cadmium-telluride detectors used in commercial IR microscopes, thereby rendering this vibration inaccessible for imaging studies. The current study reports the first IR micro-spectroscopy spectra of human iliac crest cross sections in the nu(4) PO(4)(3-) spectral regions, obtained with a synchrotron radiation source and a Cu-doped Ge detector coupled to an IR microscope. The acid phosphate (HPO(4)(2-)) content and mineral crystallite perfection (crystallinity) of a human osteon were mapped. To develop spectra-structure correlations, a combination of X-ray powder diffraction data and conventional Fourier transform IR spectra have been obtained from a series of synthetic hydroxyapatite crystals and natural bone powders of various species and ages. X-ray powder diffraction data demonstrate that there is an increase in average crystal size as bone matures, which correlates with an increase in the nu(4) PO(4)(3-) FTIR absorption peak ratio of two peaks (603/563 cm(-1)) within the nu(4) PO(4)(3-) contour. Additionally, the IR results reveal that a band near 540 cm(-1) may be assigned to acid phosphate. This band is present at high concentrations in new bone, and decreases as bone matures. Correlation of the nu(4) PO(4)(3-) contour with the nu(2) CO (3)(2-) contour also reveals that when acid phosphate content is high, type A carbonate content (i.e., carbonate occupying OH(-) sites in the hydroxyapatite lattice) is high. As crystallinity increases and acid phosphate content decreases, carbonate substitution shifts toward occupation of PO(4)(3-) sites in the hydroxyapatite lattice. Thus, IR microscopic analysis of the nu(4) PO(4)(3-) contour provides a straightforward index of both relative mineral crystallinity and acid phosphate concentration that can be applied to in situ IR micro-spectroscopic analysis of bone samples, which are of interest for understanding the chemical mechanisms of bone deposition in normal and pathological states.     

Kinetics and thermodynamics of mandelate racemase catalysis

Mandelate racemase (EC 5.1.2.2) from Pseudomonas putida catalyzes the interconversion of the two enantiomers of mandelic acid with remarkable proficiency, producing a rate enhancement exceeding 15 orders of magnitude. The rates of the forward and reverse reactions catalyzed by the wild-type enzyme and by a sluggish mutant (N197A) have been studied in the absence and presence of several viscosogenic agents. A partial dependence on relative solvent viscosity was observed for values of kcat and kcat/Km for the wild-type enzyme in sucrose-containing solutions. The value of kcat for the sluggish mutant was unaffected by varying solvent viscosity. However, sucrose did have a slight activating effect on mutant enzyme efficiency. In the presence of the polymeric viscosogens poly(ethylene glycol) and Ficoll, no effect on kcat or kcat/Km for the wild-type enzyme was observed. These results are consistent with both substrate binding and product dissociation being partially rate-determining in both directions. The viscosity variation method was used to estimate the rate constants comprising the steady-state expressions for kcat and kcat/Km. The rate constant for the conversion of bound (R)-mandelate to bound (S)-mandelate (k2) was found to be 889 +/- 40 s(-1) compared with a value of 654 +/- 58 s(-1) for kcat in the same direction. From the temperature dependence of Km (shown to equal K(S)), k2, and the rate constant for the uncatalyzed reaction [Bearne, S. L., and Wolfenden, R. (1997) Biochemistry 36, 1646-1656], we estimated the enthalpic and entropic changes associated with substrate binding (DeltaH = -8.9 +/- 0.8 kcal/mol, TDeltaS = -4.8 +/- 0.8 kcal/mol), the activation barrier for conversion of bound substrate to bound product (DeltaH# = +15.4 +/- 0.4 kcal/mol, TDeltaS# = +2.0 +/- 0.1 kcal/mol), and transition state stabilization (DeltaH(tx) = -22.9 +/- 0.8 kcal/mol, TDeltaS(tx) = +1.8 +/- 0.8 kcal/mol) during mandelate racemase-catalyzed racemization of (R)-mandelate at 25 degrees C. Although the high proficiency of mandelate racemase is achieved principally by enthalpic reduction, there is also a favorable and significant entropic contribution.     

Engineering of polyhydroxyalkanoate synthase by Ser477X/Gln481X saturation mutagenesis for efficient production of 3-hydroxybutyrate-based copolyesters

Class II polyhydroxyalkanoate synthase from Pseudomonas sp. 61-3 (PhaC1_Ps) synthesizes 3-hydroxybutyrate (3HB)-based copolyesters, P[3HB-co-3-hydroxyalkanoate (3HA)]. Four sites (130, 325, 477, and 481) in PhaC1_Ps that affect the cellular content and 3HB fraction of P(3HB-co-3HA) produced have been identified. Simple combination of beneficial mutations at the sites successfully increased 3HB fraction in the copolymers (62 mol.%). However, polymer content was often largely decreased (0.2 wt.%) regardless of an enhancement in 3HB fraction, compared to the wild-type enzyme (14 mol.% 3HB and 12 wt.%; Matsumoto et al. (2006) Biomacromolecules, 7:2436–2442). In the present study, we attempted to explore residues combination at the four sites to overcome the problem. Here, pairwise saturation mutagenesis at the neighboring sites 477 and 481 of PhaC1_Ps was performed using single and double mutations at sites 130 and 325 as templates to increase 3HB fraction in the copolymer without reducing the polymer content in recombinant Escherichia coli. These useful PhaC1_Ps mutants were screened based on enhanced P(3HB) content and were subsequently applied to P(3HB-co-3HA) production. Among the mutants tested, the Ser325Cys/Ser477Lys/Gln481Leu mutant exhibited increased 3HB fraction in copolymer (63 mol.%) and also polymer content (18 wt.%), indicating that mutation scrambling was effective for obtaining the desired mutants.     

Properties of mixtures of cholesterol with phosphatidylcholine or with phosphatidylserine studied by (13)C magic angle spinning nuclear magnetic resonance

The behavior of cholesterol is different in mixtures with phosphatidylcholine as compared with phosphatidylserine. In (13)C cross polarization/magic angle spinning nuclear magnetic resonance spectra, resonance peaks of the vinylic carbons of cholesterol are a doublet in samples containing 0.3 or 0.5 mol fraction cholesterol with 1-palmitoyl-2-oleoyl phosphatidylserine (POPS) or in cholesterol monohydrate crystals, but a singlet with mixtures of cholesterol and 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC). At these molar fractions of cholesterol with POPS, resonances of the C-18 of cholesterol appear at the same chemical shifts as in pure cholesterol monohydrate crystals. These resonances do not appear in samples of POPS with 0.2 mol fraction cholesterol or with POPC up to 0.5 mol fraction cholesterol. In addition, there is another resonance from the cholesterol C18 that appears in all of the mixtures of phospholipid and cholesterol but not in pure cholesterol monohydrate crystals. Using direct polarization, the fraction of cholesterol present as crystallites in POPS with 0.5 mol fraction cholesterol is found to be 80%, whereas with the same mol fraction of cholesterol and POPC none of the cholesterol is crystalline. After many hours of incubation, cholesterol monohydrate crystals in POPS undergo a change that results in an increase in the intensity of certain resonances of cholesterol monohydrate in (13)C cross polarization/magic angle spinning nuclear magnetic resonance, indicating a rigidification of the C and D rings of cholesterol but not other regions of the molecule.     

Fetal and postnatal mouse bone tissue contains more calcium than is present in hydroxyapatite

It has been shown for developing enamel and zebrafish fin that hydroxyapatite (HA) is preceded by an amorphous precursor, motivating us to examine the mineral development in mammalian bone, particularly femur and tibia of fetal and young mice. Mineral particle thickness and arrangement were characterized by (synchrotron) small-angle X-ray scattering (SAXS) combined with wide-angle X-ray diffraction (WAXD) and X-ray fluorescence (XRF) analysis. Simultaneous measurements of the local calcium content and the HA content via XRF and WAXD, respectively, revealed the total calcium contained in HA crystals. Interestingly, bones of fetal as well as newborn mice contained a certain fraction of calcium which is not part of the HA crystals. Mineral deposition could be first detected in fetal tibia at day 16.5 by environmental scanning electron microscopy (ESEM). SAXS revealed a complete lack of orientation in the mineral particles at this stage, whereas 1 day after birth particles were predominantly aligned parallel to the longitudinal bone axis, with the highest degree of alignment in the midshaft. Moreover, we found that mineral particle length increased with age as well as the thickness, while fetal particles were thicker but much shorter. In summary, this study revealed strong differences in size and orientation of the mineral particles between fetal and postnatal bone, with bulkier, randomly oriented particles at the fetal stage, and highly aligned, much longer particles after birth. Moreover, a part of the calcium seems to be present in other form than HA at all stages of development.     

Synthetic hispidin, a PKC inhibitor, is more cytotoxic toward cancer cells than normal cells in vitro

The trypanocidal activity of naturally occurring 6-(3,4-dihydroxystyryl)-4-hydroxy-2-pyrone (hispidin) prompted us to examine its cytotoxic activity toward normal and cancerous cells in culture. Hispidin synthesized in our laboratory to a high degree of purity (checked by 1H and 13C NMR spectroscopy) was shown to be cytotoxic (between 10-3 mol/L and 10-7 mol/L) toward normal human MRC-5 fibroblasts, human cancerous keratinocytes (SCL-1 cell line), and human cancerous pancreatic duct cells (Capan-1 cell line). Interestingly, addition of hispidin in three successive doses (between 10-5 mol/L and 10-7 mol/L) led to a 100-fold increase in activity with an enhanced activity on cancer cells compared to normal cells (50%). Synthetic hispidin was found to inhibit isoform of protein kinase C (IC50 of 2 × 10-6 mol/L), but not E. coli and placental type XV alkaline phosphatases. The enhanced activity of hispidin toward the cancerous cell lines is discussed.     

Phosphatidylcholine structure determines cholesterol solubility and lipid polymorphism

In the present work, we demonstrate that phosphatidylcholine with (16:1)9 acyl chains undergoes polymorphic rearrangements in mixtures with 0.6-0.8 mol fraction cholesterol. Studies were performed using differential scanning calorimetry, X-ray diffraction, cryo-electron microscopy, 31P NMR static powder patterns and 13C MAS/NMR. Mixtures of phosphatidylcholine with (16:1)9 acyl chains and 0.6 mol fraction cholesterol, after being heated to 100 degrees C, can form an ordered array with periodicity 14 nm which may be indicative of a cubic phase. Our results indicate that the formation of highly curved bilayer structures, such as those required for membrane fusion, can occur in mixtures of cholesterol with certain phosphatidylcholines that do not form non-lamellar structures in the absence of cholesterol. We also determine the polymorphic behavior of mixtures of symmetric phosphatidylcholines with cholesterol. Species of phosphatidylcholine with (20:1)11, (22:1)13 or (24:1)15 acyl chains in mixtures with 0.6-0.8 mol fraction cholesterol undergo a transition to the hexagonal phase at temperatures 70-80 degrees C. This is not the case for phosphatidylcholine with (18:1)6 acyl chains which remains in the lamellar phase up to 100 degrees C when mixed with as much as 0.8 mol fraction cholesterol. Thus, the polymorphic behavior of mixtures of phosphatidylcholine and cholesterol is not uncommon and is dependent on the intrinsic curvature of the phospholipid. Crystals of cholesterol can be detected in mixtures of all of these phosphatidylcholines at sufficiently high cholesterol mole fraction. What is unusual about the formation of these crystals in several cases is that cholesterol crystals are present in the monohydrate form in preference to the anhydrous form. Furthermore, after heating to 100 degrees C and recooling, the cholesterol crystals are again observed to be in the monohydrate form, although pure cholesterol crystals require many hours to rehydrate after being heated to 100 degrees C. Both the nature of the acyl chain as well as the mole fraction cholesterol determine whether cholesterol crystals in mixtures with the phospholipids will be in the monohydrate or in the anhydrous form.     

Adsorption of ionized and neutral pentachlorophenol to phosphatidylcholine membranes

We have studied adsorption of pentachlorophenol (PCP) to phosphatidylcholine (PC) membranes by measuring the electrophoretic mobility of multilayered lipid vesicles in PCP solutions. PC vesicles become negatively charged due to the adsorption of ionized PCP, and we have found that their zeta potential depends upon the ionic strength and pH of the aqueous suspension. We have shown that the experimental results can be adequately accounted for in terms of a two-component Langmuir-Stern-Grahame adsorption model assuming that the 'PCP adsorption sites' are occupied either by the neutral (HA) or the ionized (A-) species. The characteristics of adsorption isotherms of the PCP - PC membrane are as follows: the association constants are KA = 55,000 dm3/mol, KHA = 279,000 dm3/mol; 4.3 PC molecules make up each PCP adsorption site at saturation; the linear partition coefficients are beta HA = (15.5 +/- 0.7) x 10(-5) m and beta A = (3.0 +/- 0.3) x 10(-5) m. The properties of PCP adsorption isotherms for PC membranes predict an increased pKa value of membrane-bound PCP, which has been observed in related studies.     

Evaluation of metabolism using stoichiometry in fermentative biohydrogen

We first constructed full stoichiometry, including cell synthesis, for glucose mixed-acid fermentation at different initial substrate concentrations (0.8-6 g-glucose/L) and pH conditions (final pH 4.0-8.6), based on experimentally determined electron-equivalent balances. The fermentative bioH2 reactions had good electron closure (-9.8 to +12.7% for variations in glucose concentration and -3 to +2% for variations in pH), and C, H, and O errors were below 1%. From the stoichiometry, we computed the ATP yield based on known fermentation pathways. Glucose-variation tests (final pH 4.2-5.1) gave a consistent fermentation pattern of acetate + butyrate + large H2, while pH significantly shifted the catabolic pattern: acetate + butyrate + large H2 at final pH 4.0, acetate + ethanol + modest H2 at final pH 6.8, and acetate + lactate + trivial H2 at final pH 8.6. When lactate or propionate was a dominant soluble end product, the H2 yield was very low, which is in agreement with the theory that reduced ferredoxin (Fd(red)) formation is required for proton reduction to H2. Also consistent with this hypothesis is that high H2 production correlated with a high ratio of butyrate to acetate. Biomass was not a dominant sink for electron equivalents in H2 formation, but became significant (12%) for the lowest glucose concentration (i.e., the most oligotrophic condition). The fermenting bacteria conserved energy similarly at approximately 3 mol ATP/mol glucose (except 0.8 g-glucose/L, which had approximately 3.5 mol ATP/mol glucose) over a wide range of H2 production. The observed biomass yield did not correlate with ATP conservation; low observed biomass yields probably were caused by accelerated rates of decay or production of soluble microbial products.     

Bioluminescent assay of total bacterial contamination of raw milk]

A rapid (30-35 min) bioluminescence assay of total bacterial contamination (TBC) of raw milk was optimized. This method includes incubation of milk samples in the presence of Neonol-10 and medical purity grade pancreatin with further removal of nonbacterial ATP by filtration through a membrane filter, cell disruption by treatment with dimethyl sulfoxide, and measurement of ATP concentration in a reaction with the bioluminescent reagent Immolum. The TBC detection threshold is 0.5 x 10(5) colony-forming units (CFU) per ml milk. Coefficients of correlation between the standard plate count method and bioluminescence assay (R) and residual standard deviations (Sxy) in raw milk samples (n = 140) were 0.83 and 0.54, respectively. In sterilized milk samples artificially contaminated with pure cultures of the main representatives of milk microflora (coli-forms, Staphylococcus aureus, Streptococcus thermophilus, and Streptococcus group D), these values were 0.89-0.99 and 0.09-0.29, respectively. The specific content of ATP was found to be (0.8 +/- 0.1) x 10(-18) mol/CFU in coli-forms; (12.0 +/- 8.1) x 10(-18) mol/CFU in S. aureus; (35.2 +/- 16.9) x 10(-18) mol/CFU in S. thermophilus; and (42.5 +/- 1.3) x 10(-18) in Streptococcus group D.     

离子交换层析纯化透明质酸

考察6种离子交换树脂的静态吸附解析效果,选出201＊7阴离子交换树脂填柱,确定洗脱流速为0.6mL/min,40mL0.3mol/LNaCl和50mL0.5mol/L NaCl双浓度洗脱,实现透明质酸和杂蛋白的分离。制得透明质酸产品蛋白含量为0.057%,葡萄糖醛酸含量为43%,平均相对分子质量大于1.1×10^6,收率为54%,符合医用级透明质酸行业标准的要求。     

Studies on comonomer compositional distribution of bacterial poly(3-hydroxybutyrate-co-3-hydroxyhexanoate)s and thermal characteristics of their factions

The comonomer-unit compositional distributions have been investigated for bacterial poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(3HB-co-3HH)] samples with 3HH unit content of 13.8, 18.0, 22.0, and 54.0 mol %. They were comonomer compositionally fractionated using chloroform/n-heptane mixed solvent at ambient temperature. The fractionation of P(3HB-co-18.0 mol %3HH) and P(3HB-co-22.0 mol % 3HH), which could not be carried out effectively at room temperature, were refractionated at 70 degrees C in the mixed solvent. Fractions with different 3HH unit content in a wide range (from 4.4 to 80.7 mol %) were obtained. By use of these fractions with narrow compositional distribution, the comonomer composition dependence of thermal properties was investigated by differential scanning calorimetry. The melting point (T(m)) and heat of fusion (DeltaH) decreased as the 3HH unit content increased in the range of low 3HH content (<40 mol %), while they increased as the 3HH unit content increased in the high 3HH content range (>70 mol %). The minimum T(m) and DeltaH values were found to exist at 3HH unit content of about 60 mol %. The glass transition temperature (T(g)) decreased linearly with the increase of 3HH unit content. The values of T(m), DeltaH, and T(g) of P(3HB-co-3HH)s were compared with those of poly(3-hydroxybutyrate-co-3-hydroxyvalerate), poly(3-hydroxybutyrate-co-3-hydroxypropionate), and poly(3-hydroxybutyrate-co-4-hydroxybutyrate), and the effects of comonomer types on the thermal properties were revealed.