HA Production Research Articles

Ratio of intracellular precursors concentration and their flux influences hyaluronic acid molecular weight in Streptococcus zooepidemicus and recombinant Lactococcus lactis.

HA molecular weight variation in Streptococcus zooepidemicus and two recombinant Lactococcus lactis strains were investigated by chemostat experiments and metabolic flux analysis (MFA). The study showed that intracellular flux ratio of UDP-GlcUA to UDP-GlcNAc correlated directly with HA molecular weight, for all the three strains. The ratio of intracellular concentration of these HA precursors also exhibited a similar trend. Phosphoglucoisomerase activity and glucose flux towards lactic acid formation were found to be the major bottlenecks for HA production in all the three strains. The study suggests that environmental conditions and genetic manipulations that balance the intracellular flux and HA precursors concentrations will result in increased molecular weight.

Nuclear factor kappa B inhibition promotes closure of the rat ductus arteriosus (866.8)

Background: The ductus arteriosus(DA) is necessary for fetuses and closes after birth. Patent DA(PDA) often causes serious conditions in premature babies. On the other hand, some patients with congenital heart disease need PDA for living. Prostaglandin E2 (PGE2) is involved in both vasodilation and structural remodeling such as intimal thickening through cAMP pathway. However, DA remodeling is not completely prevented by inhibition of cAMP pathway, suggesting that there is a cAMP‐independent PGE2 signal pathway. Methods and Results: We found the nuclear factor kappa b (NFkB) pathway was activated more in EP4‐stimulated SMCs than in Forskolin‐treated SMCs, which was shown by differential DNA microarray analyses. The NFkB inhibitor IMD‐0354 reduced HA production in EP4‐ stimulated SMCs. DA explants from 19th fetuses were cultured for 48 hours in the presence or absence of IMD‐ 0354. IMD‐0354 treatment facilitated closure of DA explants. When IMD‐ 0354 was injected into 21st rat fetuses, the DA significantly contracted. Furthermore, IMD‐ 0354 constricted the DA dissected from 21st rat fetuses in a dose‐dependent manner. Conclusion: The present data demonstrated that NFkB inhibition facilitated DA closure. NFkB may play a role in a cAMP‐independent PGE2 signal pathway of the rat DA.

Augmentation of integrin-mediated mechanotransduction by hyaluronic acid

Changes in tissue and organ stiffness occur during development and are frequently symptoms of disease. Many cell types respond to the stiffness of substrates and neighboring cells in vitro and most cell types increase adherent area on stiffer substrates that are coated with ligands for integrins or cadherins. In vivo cells engage their extracellular matrix (ECM) by multiple mechanosensitive adhesion complexes and other surface receptors that potentially modify the mechanical signals transduced at the cell/ECM interface. Here we show that hyaluronic acid (also called hyaluronan or HA), a soft polymeric glycosaminoglycan matrix component prominent in embryonic tissue and upregulated during multiple pathologic states, augments or overrides mechanical signaling by some classes of integrins to produce a cellular phenotype otherwise observed only on very rigid substrates. The spread morphology of cells on soft HA-fibronectin coated substrates, characterized by formation of large actin bundles resembling stress fibers and large focal adhesions resembles that of cells on rigid substrates, but is activated by different signals and does not require or cause activation of the transcriptional regulator YAP. The fact that HA production is tightly regulated during development and injury and frequently upregulated in cancers characterized by uncontrolled growth and cell movement suggests that the interaction of signaling between HA receptors and specific integrins might be an important element in mechanical control of development and homeostasis.

Plant design aspects of catalytic biosyngas conversion to higher alcohols

Although biomethanol production has attracted most of the attention in the past years, there is a current trend for the synthesis of higher alcohols (i.e. ethanol, plus C3–C4) from biomass gasification. These compounds could be used directly as fuel or fuel additives for octane or cetane number enhancement. These also serve as important intermediates for the chemical industry. In this paper a comparison is performed between the different process configurations a higher alcohols production plant from biomass gasification can take. These options are modelled in Aspenplus™; all steps and important unit operations are presented with the aim to correctly evaluate the peripheral energy requirements and conclude with the overall thermodynamic limitations of the processes. The differentiation between black liquor and solid biomass gasification, the type of catalyst employed, and the effect of the recycling scheme adopted for the reutilization of unreacted syngas are evaluated. The design has to cope with the limited yields and poor selectivity of catalysts developed so far. The gas cleaning is different depending on the different requirements of the catalysts as far as H2S purity. The process modelling results reveal that the hydrogenation of CO to higher alcohols is favoured by high pressure, temperature around 325 °C and high reactor residence times. A biorefinery using modified Fisher–Tropsch (FT) catalysts (MoS2) prevail over modified MeOH catalyst (Cu–Zn based) for HA production. The efficiency of HA production in HHV terms can reach up to 25%.

Synthesis and Characterization of Hyaluronic Acid for Biomedical Application

Hyaluronic acid is a linear polysaccharide composed of glucuronic acid and N-acetylglucosamine. HA-based biomaterials secure wide-range applications in the healthcare industry. The material is found in cartilage, skin, umbilical cord, synovial fluid, as well as in the cell wall of bacteria such a Streptococcus zooepidemicus. As a consequence of these wide applications, HA production from microbial fermentation is receiving increased attention for avoiding the risk of cross-species viral infection, to have lower production cost and to favor a more efficient purification. The molecular weight is increased by adding poly-γ-glutamic acid, which inhibits hyaluronidase of medium, and the degree of purity is enhanced by a purification process using the means of dialysis membrane and organic solvent.

Preparation of Hyaluronan-DNA Matrices and Films

Natural carbohydrate is a class of underexplored polymers for gene delivery. The noninflammatory and nonimmunogenic properties of hyaluronan (hyaluronic acid, HA) are important in clinical situations. It has a role in wound repair and has great lubricating ability. Moreover, the presence of hyaluronidase in vivo enables any vehicle fabricated from HA to be degraded by enzyme-mediated erosion. When DNA is entrapped in a cross-linked HA vehicle, HA-DNA fragments are released on digestion by hyaluronidase. These fragments could serve both as microcarriers of DNA and its protective mechanism. This protocol describes preparation of water-insoluble HA-DNA matrices and films designed for clinical applications, and assays for verification of their bioactivities. Plasmid DNA (pDNA) encoding platelet-derived growth factor (PDGF) is coupled to the matrices that could be implanted into chronic wounds to accelerate their healing. pDNA encoding hyaluronan synthase 2 (HAS2) is coupled to the film that could initially serve as a physical barrier and subsequently a pDNA reservoir for sustaining HAS2 transfection. This would lead to continual HA production for preventing postsurgical adhesion.

O‐GlcNAcylation and hyaluronan synthesis

The intracellular regulation of HA synthesis throughout O‐GlcNacylation is investigated. It was reported that the HA precursor UDP‐N‐acetylglucosamine (UDP‐GlcNac) and UDP glucuronic acid availability modulates of HA synthesis. In this study, we tested the role of O‐GlcNacylation, a type of cytoplasmic protein O‐glycosylation to ser/thr residues and its influence on hyaluronan synthase activity. We found that the inhibition of OGlcNacylation strongly reduced HA production whereas treatments that induced protein O‐GlcNacylation increased HA secretion. Gene expression studies revealed that HAS2 mRNA (i.e., the main HA synthesizing enzyme located in the plasma membrane) was the most sensible to O‐GlcNacylation and accumulated after its induction. We found evidences that the transcription regulator YY1 activates HAS2 expression after O‐GlcNacylation. At protein level, experiments with wheat germ agglutinin and recombinant 6myc‐HAS2 revealed that this enzyme was actually O‐GlcNacylated. Further, HAS2 O‐GlcNacylation increased HAS activity in purified microsomes leading to HA accumulation. Interestingly, the O‐GlcNacylation was able to delay the protein removal from cell membrane increasing the enzyme turn‐over, another way to induce increase of HA. This work was supported by FAR and Onlus Varesotto‐ITALY.

Characterization of Modified Fischer–Tropsch Catalysts Promoted with Alkaline Metals for Higher Alcohol Synthesis

Two series of Cu/Co/Cr modified Fischer–Tropsch catalyst promoted with Zn or Mn and an alkaline metal (Me: Li, Na, K, Rb, Cs) were prepared by co-precipitation method and tested for high alcohol synthesis (HAS) at one hour on-stream and at two temperatures, 300 and 350 °C. The results indicate that the best selectivity toward high alcohols depends on temperature and catalysts composition and is obtained as follows: a) at 300 °C over catalysts without Zn and containing K, Na and Rb; b) at 350 °C over catalysts without Zn and containing K; c) at 350 °C over catalysts containing Zn as well as Li and Cs. Two series of Cu/Co/Cr modified Fischer–Tropsch catalyst promoted with Zn or Mn and an alkaline metal (Me: Li, Na, K, Rb, Cs) were prepared by co-precipitation method and tested for HAS at one hour on-stream and at two temperatures, 300 and 350 °C. The results indicate that the higher HA production (i.e. normal and branched butanol) is obtained at 350 °C over catalysts promoted with Zn and all alkali metals, except for Li which shown its best performance at 300 °C.

Culture Conditions Optimization of Hyaluronic Acid Production Based on GP and QPSO Algorithms

This study aimed to optimize the culture conditions (agitation speed, aeration rate and stirrer number) of hyaluronic acid production by Streptococcus zooepidemicus. Two optimization algorithms were used for comparison: response surface methodology (RSM) and genetic programming coupling Quantum-behaved particle swarm optimization algorithm (GP-QPSO). In GP -QPSO approach, GP is employed to model the microbial HA production and QPSO algorithm is used to find out the optimal culture conditions with the established GP estimator as the objective function. The maximum predicted value of HA production by RSM and GP-QPSO was 5.27 and 5.57 g/l, respectively. Here though both RSM and GP-QPSO approach provided good predictions, yet the proposed GP-QPSO method showed a clear superiority over RSM for both data fitting and optimization capabilities

ROLE OF THE HYALURONAN-PRODUCING TENOSYNOVIUM IN PREVENTING ADHESION FORMATION DURING HEALING OF FLEXOR TENDON INJURIES

Flexor tendons of white Leghorn chickens (n = 25) were used for this study. One chicken was used as a normal control (no surgery), and the remaining 24 were used for experiments. After partial tendon-severing in both legs of 24 chickens, the right and the left leg were treated differently, thereby creating two groups: Group I, in which the tenosynovium was preserved, and Group II, in which the tenosynovium was removed. Hematoxylin-eosin staining was performed to observe adhesions; immunohistochemical analysis was used to localize HA. HA production was noted in granulation tissue invading between the tendon stumps in both groups; however, HA expression in the tenosynovium was observed only in Group I where adhesion formation was minimal. The HA-producing tenosynovium plays a crucial role in preventing adhesion formation in this model of flexor tendon injuries.

Microbial production of hyaluronic acid: current state, challenges, and perspectives

Hyaluronic acid (HA) is a natural and linear polymer composed of repeating disaccharide units of β-1, 3-N-acetyl glucosamine and β-1, 4-glucuronic acid with a molecular weight up to 6 million Daltons. With excellent viscoelasticity, high moisture retention capacity, and high biocompatibility, HA finds a wide-range of applications in medicine, cosmetics, and nutraceuticals.Traditionally HA was extracted from rooster combs, and now it is mainly produced via streptococcal fermentation. Recently the production of HA via recombinant systems has received increasing interest due to the avoidance of potential toxins. This work summarizes the research history and current commercial market of HA, and then deeply analyzes the current state of microbial production of HA by Streptococcus zooepidemicus and recombinant systems, and finally discusses the challenges facing microbial HA production and proposes several research outlines to meet the challenges.

Preparation of bovine hydroxyapatite by transferred arc plasma

Hydroxyapatite (Ca 10(PO 4) 6(OH) 2, abbreviated as HA) is a kind of bioactive material that has received considerable attention over the past decades as an implant material due to its excellent biocompatability for clinical applications. In this work, Hydroxyapatite was obtained by plasma processing of the natural bovine bones by Transferred arc plasma (TAP) processing at 5 kW in argon plasma for different processing times (i.e. 30, 45, 60, 90 and 120 s). The TAP synthesized HA bioceramic was characterized by XRD, FTIR, SEM-EDX and TG-DTA analysis. The effect of TAP processing time on the preparation of organic free HA from bovine bone was studied. The study indicated that TAP processing for 30, 45 and 60 s were insufficient for removal of organics from the natural bovine bone. Organic free bovine HA was obtained for 90 s TAP processing with a Ca/P ratio of 1.93 comparable with commercially available natural HA-Endobon powder. Whereas 120 s of processing resulted in trivial thermal decomposition of HA in to its constituent phases. Thus our present investigation suggested that HA production from bovine bone using TAP processing is a time effective advantageous method in comparison to the annealing method.

High-Yield Hypocrellin A Production in Solid-State Fermentation by Shiraia sp. SUPER-H168

Hypocrellin A production by Shiraia sp. SUPER-H168 was studied under solid-state fermentation. Corn was found to be the best substrate after evaluating eight kinds of agro-industrial crops and residues. The optimized solid-state fermentation conditions were as follows: inoculum size 3 x 10(6) spores, substrate particle size 0.8-1 mm, initial moisture content 50%, and temperature 30 degrees C. Six kinds of external carbon source and seven kinds of external nitrogen source were evaluated, respectively, for HA production. Glucose and NaNO(3) were the best. The combination of them was optimized by the response surface method. The optimum compositions of the supplementary glucose and NaNO(3) were 1.65 g/100 g and 0.43 g/L, respectively. Hypocrellin A production reached 4.7 mg/g.

Culture conditions optimization of hyaluronic acid production by Streptococcus zooepidemicus based on radial basis function neural network and quantum-behaved particle swarm optimization algorithm

This study aimed to optimize the culture conditions (agitation speed, aeration rate and stirrer number) of hyaluronic acid production by Streptococcus zooepidemicus. Two optimization algorithms were used for comparison: response surface methodology (RSM) and radial basis function neural network coupling quantum-behaved particle swarm optimization algorithm (RBF-QPSO). In RBF-QPSO approach, RBF is employed to model the microbial HA production and QPSO algorithm is used to find the optimal culture conditions with the established RBF estimator as the objective function. The predicted maximum HA yield by RSM and RBF-QPSO was 5.27 and 5.62 g/l, respectively, while a maximum HA yield of 5.21 and 5.58 g/l was achieved in the validation experiments under the optimal culture conditions obtained by RSM and RBF-QPSO, respectively. It was indicated that both models provided similar quality predictions for the above three independent variables in terms of HA yield, but RBF model gives a slightly better fit to the measured data compared to RSM model. This work shows that the combination of RBF neural network with QPSO algorithm has good predictability and accuracy for bioprocess optimization and may be helpful to the other industrial bioprocesses optimization to improve productivity.

Comparison of Hydroxyapatite and AB-Type Carbonate-Substituted Hydroxyapatite Suspensions for Use in the Reticulated Foam Method of Scaffold Production

Precipitation routes for HA production result in the formation of suspensions of nano-scale hydroxyapatite particles in water. During this work, suspensions of phase-pure and carbonate-substituted hydroxyapatite were produced using a precipitation reaction with calcium hydroxide and orthophosphoric acid as reactants. The chemistry of the apatites was analysed using XRD and FTIR after heat treatment in various atmospheres and the particle morphology investigated using TEM. The thermal stability of the carbonate HA was found to be significantly less than the phase-pure HA and dependent upon the heat-treatment atmosphere. Suspensions were used directly for replication of polymer foam templates with a dip-coating technique. Good replication of the foam structure was achieved with the phase pure hydroxyapatite, but no structural integrity was achieved with the carbonate-HA structure possibly a result of limits on the sintering temperature.

Isolation and identification of a new hypocrellin A-producing strain Shiraia sp. SUPER-H168

A new hypocrellin A-producing strain, Shiraia sp. SUPER-H168, was isolated from tissues of bamboo, Brachystachyum densiflorum. The morphology of this strain was characterized with a light microscope and a scanning electronic microscope. The mycelia, conidia, pycnidia of fungus were observed. Small pycnidia (10-20 microm in length) full of small conidia appeared on the mycelia, which were first reported in this study. The 18S rDNA region of this strain was amplified and sequenced. Then a neighbor-joining tree of 18S rDNA was constructed. According to the result of analysis, the strain SUPER-H168 was proved to belong to the genus Shiraia. Hypocrellin A was produced by solid-state fermentation, extracted by acetone, isolated by preparative RP-HPLC, and identified by RP-HPLC, ESI-MS and ultraviolet-visible absorbing scanning with diode array detection. The HA production could reach 2.02 mg/g dry solid substrate.

Hyaluronic Acid Synthase-1 Expression Regulates Bladder Cancer Growth, Invasion, and Angiogenesis through CD44

Hyaluronic acid (HA) promotes tumor metastasis and is an accurate diagnostic marker for bladder cancer. HA is synthesized by HA synthases HAS1, HAS2, or HAS3. We have previously shown that HAS1 expression in tumor tissues is a predictor of bladder cancer recurrence and treatment failure. In this study, we stably transfected HT1376 bladder cancer cells with HAS1-sense (HAS1-S), HAS1-antisense (HAS1-AS), or vector cDNA constructs. Whereas HAS1-S transfectants produced approximately 1.7-fold more HA than vector transfectants, HA production was reduced by approximately 70% in HAS1-AS transfectants. HAS1-AS transfectants grew 5-fold slower and were approximately 60% less invasive than vector and HAS1-S transfectants. HAS1-AS transfectants were blocked in G(2)-M phase of the cell cycle due to down-regulation of cyclin B1, cdc25c, and cyclin-dependent kinase 1 levels. These transfectants were also 5- to 10-fold more apoptotic due to the activation of the Fas-Fas ligand-mediated extrinsic pathway. HAS1-AS transfectants showed a approximately 4-fold decrease in ErbB2 phosphorylation and down-regulation of CD44 variant isoforms (CD44-v3, CD44-v6, and CD44-E) both at the protein and mRNA levels. However, no decrease in RHAMM levels was observed. The decrease in CD44-v mRNA levels was not due to increased mRNA degradation. Whereas CD44 small interfering RNA (siRNA) transfection decreased cell growth and induced apoptosis in HT1376 cells, HA addition modestly increased CD44 expression and cell growth in HAS1-AS transfectants, which could be blocked by CD44 siRNA. In xenograft studies, HAS1-AS tumors grew 3- to 5-fold slower and had approximately 4-fold lower microvessel density. These results show that HAS1 regulates bladder cancer growth and progression by modulating HA synthesis and HA receptor levels.

Chronic activation of the prostaglandin receptor EP4 promotes hyaluronan-mediated neointimal formation in the ductus arteriosus

PGE, a potent vasodilator, plays a primary role in maintaining the patency of the ductus arteriosus (DA). Genetic disruption of the PGE-specific receptor EP4, however, paradoxically results in fatal patent DA (PDA) in mice. Here we demonstrate that EP4-mediated signals promote DA closure by hyaluronic acid-mediated (HA-mediated) intimal cushion formation (ICF). Chronic EP4 stimulation by ONO-AE1-329, a selective EP4 agonist, significantly enhanced migration and HA production in rat DA smooth muscle cells. When HA production was inhibited, EP4-mediated migration was negated. Activation of EP4, adenylyl cyclase, and PKA all increased HA production and the level of HA synthase 2 (HAS2) transcripts. In immature rat DA explants, ICF was promoted by EP4/PKA stimuli. Furthermore, adenovirus-mediated Has2 gene transfer was sufficient to induce ICF in EP4-disrupted DA explants in which the intimal cushion had not formed. Accordingly, signals through EP4 have 2 essential roles in DA development, namely, vascular dilation and ICF. The latter would lead to luminal narrowing, helping adhesive occlusion and permanent closure of the vascular lumen. Our results imply that HA induction serves as an alternative therapeutic strategy for the treatment of PDA to the current one, i.e., inhibition of PGE signaling by cyclooxygenase inhibitors, which might delay PGE-mediated ICF in immature infants.

Osteopontin Induction of Hyaluronan Synthase 2 Expression Promotes Breast Cancer Malignancy

Osteopontin (OPN) is a tumor-associated, secreted phosphoprotein that has been implicated in breast cancer progression and metastasis. Research concerning how OPN functions in tumor progression has led to the identification of a limited number of genes that contribute functionally to OPN-induced cellular behaviors. Recent microarray analysis, comparing 21NT breast cancer cells transfected to constitutively overexpress OPN with control cells, revealed hyaluronan synthase 2 (HAS2) to be a gene highly up-regulated in OPN-overexpressing cells. In this study, we further examined the relationship between OPN and HAS2. We show that 21NT OPN-transfected cells express high levels of HAS2, which is associated with increased HA production and matrix retention and is necessary for tumor cell adhesion to bone marrow endothelial cells and anchorage-independent growth. Finally, stable transfection of antisense HAS2 into 21NT cells overexpressing OPN resulted in a reduction in HAS2 expression, HA production, and pericellular retention. Antisense-mediated down-regulation of HAS2 also resulted in a significant decrease in cellular proliferation and colony growth in soft agar. To our knowledge, this is the first report of the ability of OPN to regulate HAS2 expression and HA production in breast cancer cells and further illustrates a unique functional relationship by which enhanced HA production facilitates OPN-mediated cell behaviors.

The properties of recombinant Sendai virus having the P gene of Sendai virus pi strain derived from BHK cells persistently infected with Sendai virus

We prepared the chimeric recombinant Sendai virus [rSeV(Ppi)] by replacing the P gene of the Z strain with that of pi strain for analyzing the function of Ppi, Vpi and Cpi proteins. Intriguingly, HA production by rSeV(Ppi) is significantly lower at 38 degrees C than at 32 degrees C, showing that virus growth of rSeV(Ppi) is slightly suppressed at 38 degrees C. However, the main phenotypes of SeVpi, a marked temperature sensitivity as viral replication and an ability of establishing persistent infection, are not explained by the Ppi, Vpi and Cpi proteins. The V and C proteins form inclusion bodies in L929 cells infected with rSeV(Ppi) and incubated at 38 degrees C. L929 cells infected with rSeV(Ppi) and L929 cells stably expressing the Cpi protein show resistance to interferon-beta at 32 and 38 degrees C, indicating that the Cpi protein per se is not temperature-sensitive to inhibition of IFN signaling. The complete genome sequences of Sendai virus (SeV) pi and parent Nagoya strains were determined. Fifty nucleic acid substitutions are found in the genome sequence of SeV pi strain in comparison with Nagoya strain. There are three nucleic acid substitutions in the leader sequence, while the trailer, intergenic, gene-end and gene-start sequences of both strains are completely identical. Deletions and insertions of nucleotide are not found. There are 32 amino acid substitutions in Sendai virus pi strain. The specific amino acid substitutions unique to the SeVpi are 18. Information about the complete genome sequences of SeVpi strain is important to totally understand the persistent infection and lower pathogenicity of SeV.