The effects of herring-roe lyophilized powder on lipid metabolism.

Herring-roe, which contains large amounts of docosahexaenoic acid and eicosapentaenoic acid, has anti-dyslipidemia effects. Here, we evaluated the effects of herring-roe on lipid metabolism in 33 adult subjects in a randomized, double-blind, placebo-controlled study. We divided the subjects into a test group that ingested herring-roe lyophilized powder (herring-roe powder) and a placebo group that ingested non-herring-roe powder, with each member of each group ingesting 15 g daily for 8 weeks. Hematological tests and body composition measurements were performed before and after 4, 6, and 8 weeks of the study period. Although no significant differences in low density lipoprotein were observed, high density lipoprotein was found to be increased in subjects who ingested herring-roe powder. In addition, the level of free fatty acid was significantly improved in the herring-roe powder group. These results suggest that ingestion of herring-roe could influence lipid metabolism.

Engineering of L-tyrosine oxidation in Escherichia coli and microbial production of hydroxytyrosol.

The hydroxylation of tyrosine is an important reaction in the biosynthesis of many natural products. The use of bacteria for this reaction has not been very successful due to either the over-oxidation to ortho-quinone when using tyrosinases from bacteria or plants, or the lack of the native cofactor, tetrahydrobiopterin (BH4), needed for the activity of tyrosine hydroxylases (TH). Here, we demonstrate that an Escherichia coli cofactor, tetrahydromonapterin (MH4), can be used as an alternative cofactor for TH in presence of the BH4 regeneration pathway, and tyrosine hydroxylation is performed without over-oxidation. We used this platform for biosynthesis of one of the most powerful antioxidants, hydroxytyrosol. An endogenous aromatic aldehyde oxidase was identified and knocked out to prevent formation of the side product, and this resulted in nearly exclusive production of hydroxytyrosol in engineered E. coli. Finally, hydroxytyrosol production from a simple sugar as a sole carbon source was demonstrated.

Engineering of a tyrosol-producing pathway, utilizing simple sugar and the central metabolic tyrosine, in Escherichia coli.

Metabolic engineering was applied to the development of Escherichia coli capable of synthesizing tyrosol (2-(4-hydroxyphenyl)ethanol), an attractive phenolic compound with great industrial value, from glucose, a renewable carbon source. In this strain, tyrosine, which was supplied not only from the culture medium but also from the central metabolism, was converted into tyrosol via three steps: decarboxylation, amine oxidation, and reduction. The engineered strain synthesized both tyrosol and 4-hydroxyphenylacetate (4HPA), but disruption of the endogenous phenylacetaldehyde dehydrogenase gene shut off 4HPA production and improved the production of tyrosol as a sole product. The engineered mutant strain was capable of producing 0.5 mM tyrosol from 1% (w/v) glucose during a 48 h shake flask cultivation.

In vitro synthesis of polyhydroxyalkanoate (PHA) incorporating lactate (LA) with a block sequence by using a newly engineered thermostable PHA synthase from Pseudomonas sp. SG4502 with acquired LA-polymerizing activity.

Recently, we succeeded in isolating a thermotolerant bacterium, Pseudomonas sp. SG4502, which is capable of accumulating polyhydroxyalkanoate (PHA) even at 55 °C, as a source of thermostable enzymes. In this study, we cloned a pha locus from the bacterium and identified two genes encoding PHA synthases (PhaC1(SG) and PhaC2(SG)). Two mutations, Ser324Thr and Gln480Lys, corresponding to those of a lactate (LA)-polymerizing enzyme (LPE) from mesophilic Pseudomonas sp. 61-3 were introduced into PhaC1(SG) to evaluate the potential of the resulting protein as a "thermostable LPE". The mutated PhaC1(SG) [PhaC1(SG)(STQK)] showed high thermal stability in synthesizing P(LA-co-3HB) in an in vitro reaction system under a range of high temperatures. Requirement of 3HBCoA as a priming unit for LA polymerization by the LPE has been suggested in both of the in vitro and in vivo experiments. Based on the finding, the PhaC1(SG)(STQK)-mediated synthesis of a LA-based copolymer with a block sequence was achieved in the in vitro system by sequential feeding of the corresponding two substrates. This in vitro reaction system using the thermostable LPE provides us with a versatile way to synthesize the various types of LA-based copolymers with desired sequence patterns, random or block, depending on the way of supplying hydroxyalkanoates (mixed or sequential feeding).

Chemo-enzymatic synthesis of polyhydroxyalkanoate (PHA) incorporating 2-hydroxybutyrate by wild-type class I PHA synthase from Ralstonia eutropha.

A previously established improved two-phase reaction system has been applied to analyze the substrate specificities and polymerization activities of polyhydroxyalkanoate (PHA) synthases. We first analyzed the substrate specificity of propionate coenzyme A (CoA) transferase and found that 2-hydroxybutyrate (2HB) was converted into its CoA derivative. Then, the synthesis of PHA incorporating 2HB was achieved by a wild-type class I PHA synthase from Ralstonia eutropha. The PHA synthase stereoselectively polymerized (R)-2HB, and the maximal molar ratio of 2HB in the polymer was 9 mol%. The yields and the molecular weights of the products were decreased with the increase of the (R)-2HB concentration in the reaction mixture. The weight-average molecular weight of the polymer incorporating 9 mol% 2HB was 1.00 × 10(5), and a unimodal peak with polydispersity of 3.1 was observed in the GPC chart. Thermal properties of the polymer incorporating 9 mol% 2HB were analyzed by DSC and TG-DTA. T (g), T (m), and T (d) (10%) were observed at -1.1°C, 158.8°C, and 252.7°C, respectively. In general, major components of PHAs are 3-hydroxyalkanoates, and only engineered class II PHA synthases have been reported as enzymes having the ability to polymerize HA with the hydroxyl group at C2 position. Thus, this is the first report to demonstrate that wild-type class I PHA synthase was able to polymerize 2HB.

In vivo effects of isolated implantation of salmon-derived crosslinked atelocollagen sponge into an osteochondral defect.

We have developed crosslinked salmon-derived atelocollagen (SC) sponge, which has a denaturation temperature of 47°C. Sixty-four knees of 32 mature rabbits were randomly divided into 4 groups after creating an osteochondral defect in the femoral trochlea. Defects in Groups I, II, and III were filled with the crosslinked SC sponge, the crosslinked porcine collagen (PC) sponge, and the non-crosslinked PC sponge, respectively. In Group IV, defects were left untreated as the control. At 12 weeks after implantation, the histological score showed that Group I was significantly greater than Groups III (P = 0.0196) and IV (P = 0.0021). In addition, gene expression of type-2 collagen, aggrecan, and SOX9 was the greatest in Group I at 12 weeks. The fundamental in vivo properties of the crosslinked SC sponge showed that this is a promising biomaterial, specifically as a scaffold for cartilage tissue engineering.

Structure of bacterial cellulose synthase subunit D octamer with four inner passageways.

The cellulose synthesizing terminal complex consisting of subunits A, B, C, and D in Acetobacter xylinum spans the outer and inner cell membranes to synthesize and extrude glucan chains, which are assembled into subelementary fibrils and further into a ribbon. We determined the structures of subunit D (AxCeSD/AxBcsD) with both N- and C-terminal His(6) tags, and in complex with cellopentaose. The structure of AxCeSD shows an exquisite cylinder shape (height: ∼65 Å, outer diameter: ∼90 Å, and inner diameter: ∼25 Å) with a right-hand twisted dimer interface on the cylinder wall, formed by octamer as a functional unit. All N termini of the octamer are positioned inside the AxCeSD cylinder and create four passageways. The location of cellopentaoses in the complex structure suggests that four glucan chains are extruded individually through their own passageway along the dimer interface in a twisted manner. The complex structure also shows that the N-terminal loop, especially residue Lys6, seems to be important for cellulose production, as confirmed by in vivo assay using mutant cells with axcesD gene disruption and N-terminus truncation. Taking all results together, a model of the bacterial terminal complex is discussed.

Chemo-enzymatic synthesis of polyhydroxyalkanoate by an improved two-phase reaction system (TPRS).

In our previous paper, we synthesized poly-3-hydroxybutyrate [P(3HB)] by using the water-organic solvent two-phase reaction system (TPRS), in which thiophenyl (R)-3-hydroxybutyrate [(R)-3HBTP] was used as a precursor of 3HBCoA. We have developed an improved TPRS for the chemo-enzymatic synthesis of polyhydroxyalkanoate (PHA). In this method, acetyl thioester of ethyl thioglycolate (AcETG) was used as a precursor of acetylCoA (AcCoA), which was a donor of CoA. The AcCoA was formed by the ester exchange reaction between CoA in the water phase and AcETG in the organic solvent phase. The AcCoA and free 3-hydroxybutyrate (3HB) in the water phase were converted into 3HBCoA and acetate by a CoA transfer reaction of propionylCoA transferase (PCT). The synthesized 3HBCoA was polymerized sequentially by PHA synthase, and P(3HB) was successfully formed. The maximal yield of P(3HB) was 1.2 g/l under the optimal reaction condition; this is comparable to that of in vivo PHA production. Furthermore, the number of enzymes was reduced and enzyme preparation was simplified by the construction of a fusion protein, PCT-PhaC. The chemo-enzymatic synthesis of P(3HB-co-3-hydroxypropionate) and P(3HB-co-3-mercaptopropionate) was also achieved by the improved TPRS using the fusion protein.

Development of tissue-engineered human periodontal ligament constructs with intrinsic angiogenic potential.

One approach to treat periodontal diseases is grafting of tissue-engineered periodontal ligaments. Therefore, periodontal ligaments were constructed by layering cell sheets. A cell sheet was prepared by enzymatic digestion of salmon collagen gel on which human periodontal ligament fibroblasts (HPLFs) were co-cultured with or without human umbilical vein endothelial cells (HUVECs). Three cell sheets were layered and then cultured in angiogenic media, in which the HUVECs were found to form capillary-like structures when co-cultured on the HPLFs. The layered HPLFs sheets with HUVEC co-culture (PL-EC construct) demonstrated longer survival, higher alkaline phosphatase activities and lower osteocalcin production than layered HPLFs sheets without HUVEC co-culture (PL construct). Hematoxylin-eosin and Masson's trichrome staining of histological sections showed that cell density, mass and extracellular matrix deposition of the PL-EC construct were higher than those of the PL construct. Furthermore, CD31 immunostaining revealed the formation of capillary-like structures throughout the PL-EC construct. In conclusion, we successfully developed tissue-engineered periodontal ligament constructs with intrinsic angiogenic potential using cell sheet engineering and HUVEC co-culture.

Preparation and characterization of collagen from soft-shelled turtle (Pelodiscus sinensis) skin for biomaterial applications.

Collagen was isolated from the skin of soft-shelled turtle (Pelodiscus sinensis) by acid solubilization with pepsin. The yield of soft-shelled turtle collagen (STC) was 12.1% on a dry weight basis. The electrophoresis assay showed that STC consisted of a alpha(1)alpha(2) heterodimer similar to porcine collagen (PC). Amino-acid composition analysis showed that the hydroxyproline content of STC was 7.8%, which was lower than that of PC (9.5%). The denaturation temperature of STC was 36 degrees C from optical rotation analysis. An accelerated fibrillogenesis of STC was observed in phosphate-buffered saline at 25 degrees C. The resulting STC fibrillar gel had microfibrillar network with fibril diameter of ca. 124 nm, as revealed by observation with scanning electron microscopy. The compressive moduli of the STC gel and the PC gel were 3.2 +/- 0.8 kPa and 3.6 +/- 0.3 kPa, respectively. The potential of the STC gel for biomaterial applications was investigated by in vitro cell culture. Human dermal fibroblasts were three-dimensionally cultured in the STC gel and their growth was evaluated by DNA content measurement. Steady growth was observed in the STC gel for a 6-day culture period, although the growth rate was slower than in the PC gel. In conclusion, STC could be used as a novel collagen source for biomaterial applications.

Development of novel covered stents using salmon collagen.

In this study, we newly developed self-expandable or balloon-expandable covered stents with a biodegradable salmon collagen (SC) film. The SC-covered stents were fabricated by placing a bare stent in a mixture of acidic SC solution and a fibrillogenesis-inducing buffer (pH 6.8) including a cross-linking agent (water-soluble carbodiimide), and subsequent incubation at 4 degrees C for 24 h and lyophilization. The stents obtained were completely covered with an SC film having a nanofibrous structure (fibril diameter, about 70 nm). On immersion in water, the film is converted to a gel with slight swelling. There was no rupture of the SC cover after mounting on a balloon catheter or after expansion. Preliminary implantation was conducted by placing the balloon-expandable covered stents in the common carotid arteries of beagles. One month after implantation, angiography showed that all stented arteries were patent with no significant neointimal thickening. In conclusion, SC is potentially useful as a cover material of endovascular stents to enhance patency.

A microbial factory for lactate-based polyesters using a lactate-polymerizing enzyme.

Polylactate (PLA) is synthesized as a representative bio-based polyester by the chemo-bio process on the basis of metal catalyst-mediated chemical polymerization of lactate (LA) supplied by microbial fermentation. To establish the one-step microbial process for synthesis of LA-based polyesters, we explored whether polyhydroxyalkanoate (PHA) synthase would exhibit polymerizing activity toward a LA-coenzyme A (CoA), based on the fact that PHA monomeric constituents, especially 3-hydroxybutyrate (3HB), are structurally analogous to LA. An engineered PHA synthase was discovered as a candidate by a two-phase in vitro polymerization system previously developed. An LA-CoA producing Escherichia coli strain with a CoA transferase gene was constructed, and the generation of LA-CoA was demonstrated by capillary electrophoresis/MS analysis. Next, when the engineered PHA synthase gene was introduced into the resultant recombinant strain, we confirmed the one-step biosynthesis of the LA-incorporated copolyester, P(6 mol% LA-co-94 mol% 3HB), with a number-average molecular weight of 1.9 x 10(5), as revealed by gel permeation chromatography, gas chromatography/MS, and NMR.

Blood compatibility evaluation of elastic gelatin gel from salmon collagen.

Blood compatibility of a novel elastic gelatin gel (e-gel) from salmon collagen was evaluated. After a 10-min incubation of the e-gel with rat whole blood, there was a macroscopically small thrombus formation on the e-gel. Microscopic observation revealed that few platelets had adhered to the e-gel. Furthermore, the platelet adhesion rate was markedly lower on the e-gel compared to collagen-coated and fibrinogen-coated surfaces. Comparable results were obtained with re-crosslinked e-gel. In conclusion, the e-gel demonstrated good blood compatibility.

Biological properties of crosslinked salmon collagen fibrillar gel as a scaffold for human umbilical vein endothelial cells.

Collagen derived from chum salmon (Oncorhynchus keta) was crosslinked with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) during collagen fibrillogenesis and applied to an in vitro cell culture to evaluate its potential use as a scaffold for vascular tissue engineering. Human umbilical vein endothelial cells (HUVEC) were cultured on the crosslinked salmon collagen fibrillar gel (EDC-SC gel), and their growth rates and production levels of cytokines, including platelet-derived growth factor-BB and von Willebrand factor, were measured. Comparison was also made with bovine collagen gel crosslinked with EDC (EDC-BC gel). The growth and cytokine production of the HUVEC cultured on the EDC-SC gel were higher than those on the EDC-BC gel. In addition, HUVEC were found to attach to the EDC-BC gel through alpha2beta1 integrin for native collagen, whereas they attached to the EDC-SC gel through alphavbeta3 integrin for denatured collagen as well as the alpha2beta1 integrin, indicating that HUVEC recognized denatured domains in the EDC-SC gel. In conclusion, the EDC-SC gel can be used as a scaffold to support HUVEC growth, although the integrin-mediated attachment manner differs between the two gels.

Regulation of endoglucanase gene (cmcax) expression in Acetobacter xylinum.

Although cellulose is the most abundant biopolymer in nature, the detailed mechanisms of cellulose biosynthesis remain unknown. Acetobacter xylinum is one of the best-studied model organisms for cellulose biosynthesis. Interestingly, the over-expression of the cmcax gene cause enhancement of cellulose production in A. xylinum, while its product (CMCax) has cellulose degradation activity. The addition of CMCax into medium also promotes cellulose production, suggesting that CMCax is involved in cellulose synthetic pathway. In the present study, we reveal the regulation mechanism of cmcax expression in A. xylinum. First, we treated cells with four kinds of beta-glucodisaccharide. Using an enzyme assay and real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), we observed an increase in CMCax activity and an induction of cmcax expression by gentiobiose treatment. Therefore, we concluded that gentiobiose induced cmcax expression. Although gentiobiose does not originally exist in the cultivation medium, we have revealed that membrane and intra-cellular proteins extracted from A. xylinum produce gentiobiose from glucose, which is one of the components in the cultivation medium. Furthermore, we confirmed that cmcax expression in a wild-type strain increased gradually after 5 d cultivation using real-time qRT-PCR. These results have led us to conclude that the increase in cmcax expression after 5 d cultivation is caused by the increase in gentiobiose, which could be synthesized by a condensation reaction in A. xylinum. Since CMCax plays a pivotal role in the cellulose production system, our results will contribute to the elucidation of mechanisms of cellulose biosynthesis.

Development of salmon milt DNA/salmon collagen composite for wound dressing.

This study aims to develop a novel wound dressing comprising salmon milt DNA (sDNA) and salmon collagen (SC). The sDNA/SC composites were prepared by incubating a mixture of an acidic SC solution, an sDNA solution, and a collagen fibrillogenesis inducing buffer (pH 6.8) containing a crosslinking agent (water-soluble carbodiimide) for gelation, and a subsequent ventilation-drying process to give sDNA/SC films. The conjugation between sDNA and SC were confirmed by sDNA-elution assay and fluorescence microscopy. The sDNA/SC films with various doses of sDNA (sDNA/SC weight ratios of 1:5, 1:10, and 1:20) were used for in vitro cell cultures to evaluate their growth potentials of normal human dermal fibroblasts (NHDF) and normal human epidermal keratinocytes (NHEK). It was found that NHDF proliferation was increased by sDNA conjugation, whereas NHEK proliferation was dose-dependently inhibited. In light of the in vitro results, the appropriate dose of sDNA for in vivo study was determined to be the ratio of 1:10. For the implantation in full-thickness skin defects in rat dorsal region, the sDNA/SC films were reinforced by incorporating them on a porous SC sponge, because the sDNA/SC films exhibited early contraction and inadequate morphologic stability when implanted in vivo. The regenerated tissue in the sDNA/SC sponge group showed similar morphology to native dermis, while the SC sponge group without sDNA showed epithelial overgrowth, indicating that additional sDNA could reduce epidermal overgrowth. Furthermore, blood capillary formation was significantly enhanced in the sDNA/SC sponge group when compared to the SC sponge group. In conclusion, the results suggest that the sDNA/SC composite could be a potential wound dressing for clinical applications.

Application of elastic salmon collagen gel to uniaxial stretching culture of human umbilical vein endothelial cells.

In this study, we investigated the potential of an elastic salmon collagen gel (e-gel) for use as stretching culture scaffold. First, human umbilical vein endothelial cells (HUVECs) were cultured on the e-gel under static condition, and their growth was evaluated by DNA content measurement, MTT test, and scanning electron microscopy. The results demonstrated steady increases in cell number with culture time. Next, HUVECs were cultured on the e-gel under static condition for 2 d, then uniaxially stretched at a constant frequency (10% elongation at 1 Hz). After the stretching culture for 2 h, the cells oriented perpendicularly to the stretch direction. Moreover, the interleukin-6 and interleukin-8 productions of the cells significantly increased under the stretch condition compared with those under the static condition. These results were in good agreement with the published data in which an elastic silicone membrane was used as a scaffold. In conclusion, the e-gel can be used for stretching culture for vascular tissue engineering.

Development of salmon collagen vascular graft: mechanical and biological properties and preliminary implantation study.

Elastic salmon collagen (SC) vascular grafts were prepared by incubating a mixture of acidic SC solution and a fibrillogenesis-inducing buffer containing a crosslinking agent [water-soluble carbodiimide (WSC)] in a tubular mold at 4 degrees C for 24 h and then at 60 degrees C for 5 min. Subsequently, re-crosslinking in ethanol solution containing WSC was performed. The dimension of the SC grafts was easily controlled by changing the size of the mold used. The compliance (stiffness parameter: beta) and burst strength of the SC grafts (internal diameter, 2 mm; length, 20 mm; and wall thickness, 0.75 mm) that were prepared for implantation were 18.2 and 1434 mmHg, respectively; both these values were comparable with those of native vessels. Upon placement in rat subcutaneous pouches, the SC grafts were gradually biodegraded with little inflammatory reaction. The SC grafts were preliminarily implanted in rat abdominal aortas by using specially designed vascular connecting system. This system was used because the graft exhibited easy tearing and thus inadequate suturability. There was neither aneurysm formation nor graft rupture, but mild thrombus formation was seen within the 4-week observation period. These grafts may be ideal for use in regenerative medicine because we believe that SC would be completely replaced with native vascular tissues after implantation, although further improvement in the mechanical properties of the graft is needed for anastomosis.

Purification, crystallization and preliminary X-ray studies of AxCesD required for efficient cellulose biosynthesis in Acetobacter xylinum.

AxCesD protein required for bacterial cellulose biosynthesis in Acetobacter xylinum was overexpressed in E. coli, purified and crystallized. Single crystals of SeMet-substituted AxCesD were obtained by the sitting-drop vapor-diffusion method. The crystal belongs to the primitive trigonal space group P3 2, with unit-cell parameters a = b = 77.7 A, and c = 213.9 A. The asymmetric unit in the crystal was assumed to contain 8 protein molecules giving the Matthews coefficient (VM) of 2.54 A3 Da(-1). Se-MAD data were collected to 2.3 A resolution using synchrotron radiations.

Preparation and characterization of multilayered hydroxyapatite/silk fibroin film.

We prepared multilayered films consisting of silk fibroin (SF) and hydroxyapatite (HAp) by alternating lamination using untreated SF and HAp-deposited SF films. Untreated SF films were prepared from a regenerated SF solution by air drying. HAp-deposited SF films were prepared by soaking methanol-treated SF films containing >5 wt% CaCl2 in a simulated body fluid with the ion concentration 1.5-fold higher than that of the standard one. The multilayered HAp/SF films had HAp layers with approximate thicknesses of 3-5 microm and SF layers with thicknesses of 40-70 microm. The bonding strength between the SF and HAp layers was significantly affected by temperature and compression time under the lamination method. The optimal conditions for achieving the maximum T-peel strength and beta-sheet contents were determined to be 130 degrees C for 4 min. The Young's modulus of the multilayered films (133.4 MPa) was higher than that of the films consisting of SF alone (92.5 MPa) under swollen conditions. The biocompatibility of the HAp-deposited SF films was analyzed by culturing of osteoblasts (MC3T3-E1) on a film. The results indicate that HAp-deposited SF films and SF films show similar degrees of cell adhesion and alkaline phosphatase activities.