Production of precursors for anti-Alzheimer drugs: Asymmetric bioreduction in a packed-bed bioreactor using immobilized D. carota cells

(S)-1-Phenylethanol derivatives, which are the precursors of many pharmacological products, have also been used as anti-Alzheimer drugs. Bioreduction experiments were performed in a batch and packed-bed bioreactor. Then, the kinetics constants were determined by examining the reaction kinetics in the batch system with free and immobilized carrot cells. Also, the effective diffusion coefficient (D_e) of acetophenone in calcium alginate-immobilized carrot cells was investigated. Kinetics constants for free cells, which are intrinsic values, are reaction rate V_max?=?0.052?mmol?L^?1?min^?1, and constants of the Michaelis–Menten K_M?=?2.31?mmol?L^?1. Kinetics constants for immobilized cells, which are considered apparent values, are V_{max, app}?=?0.0407?mmol?L^?1 min^?1, K_{M, app}?=?3.0472?mmol?L^?1 for 2?mm bead diameter, and V_{max, app}?=?0.0453?mmol?L^?1 min^?1, K_{M, app}?=?4.9383?mmol?L^?1 for 3?mm bead diameter. Average value of effective diffusion coefficient of acetophenone in immobilized beads was determined as 1.97?×?10^?6?cm²?s^?1. Using immobilized carrot cells in an up-flow packed-bed reactor, continuous production of (S)-1-phenylethanol through asymmetric bioreduction of acetophenone was performed. The effects of the residence time and concentrations of substrate were investigated at pH 7.6 and 33°C. Enantiomerically pure (S)-1-phenylethanol (ee?>?99%) was produced with 75% conversion at 4-hr residence time.     

Radio-protective and antioxidative activities of astaxanthin from newly isolated radio-resistant bacterium <Emphasis Type="Italic">Deinococcus</Emphasis> sp. strain WMA-LM9

A radio-resistant bacterium, designated as strain WMA-LM9, was isolated from desert soil. 16S rRNA gene sequencing indicated that the bacterium belongs to genus Deinococcus with maximum similarity to Deinococcus radiopugnans. Deinococcus sp. strain WMA-LM9 was found to be resistant to a ultraviolet (UV) dose of 5 × 10³ J/m², hydrogen peroxide (50 mM) and mitomycin C (10 μg/ml). A carotenoid pigment was extracted using chloroform/methanol/acetone (7:5:3) and purified by high-performance liquid chromatography on a C₁₈ analytical column. The compound was characterised as mono-esterified astaxanthin by ¹H, ¹³C nuclear magnetic resonance and mass spectrometry. It was tested for antioxidant activity, total flavonoids and phenolic content, radioprotective potential in correlation to the prevention of protein oxidation and DNA strand breaks in vitro. The carotenoid pigment showed a very potent antioxidant activity and significantly stronger scavenging ability against superoxides, with an IC₅₀ (concentration causing 50% inhibition of the desired activity) of 41.6 μg/ml. The total phenolic and flavonoid contents were 12.1 and 7.4 μg in terms of gallic acid and quercetin equivalents per milligram of dried mass, respectively. astaxanthin also showed a higher inhibitory action against oxidative damage to collagen, elastin and bovine serum albumin than did β-carotene. The carotenoid also inhibited breaks to DNA strands, as indicated by the results of the DNA damage prevention assay. We conclude that astaxanthin from Deinococcus sp. strain WMA-LM9 has protective effects against radiation-mediated cell damage, and it also protects cellular protein and DNA against oxidative stress and other anti-oxidant activities.     

Insights into a key sulfite scavenger enzyme sulfite oxidase (<Emphasis Type="Italic">SOX</Emphasis>) gene in plants

Sulfite oxidase (SOX) is a crucial molybdenum cofactor-containing enzyme in plants that re-oxidizes the sulfite back to sulfate in sulfite assimilation pathway. However, studies of this crucial enzyme are quite limited hence this work was attempted to understand the SOXs in four plant species namely, Arabidopsis thaliana, Solanum lycopersicum, Populus trichocarpa and Brachypodium distachyon. Herein studied SOX enzyme was characterized with both oxidoreductase molybdopterin binding and Mo-co oxidoreductase dimerization domains. The alignment and motif analyses revealed the highly conserved primary structure of SOXs. The phylogeny constructed with additional species demonstrated a clear divergence of monocots, dicots and lower plants. In addition, to further understand the phylogenetic relationship and make a functional inference, a structure-based phylogeny was constructed using normalized RMSD values in five superposed models from four modelled plant SOXs herein and one previously characterized chicken SOX structure. The plant and animal SOXs showed a clear divergence and also implicated their functional divergences. Based on tree topology, monocot B. distachyon appeared to be diverged from other dicots, pointing out a possible monocot–dicot split. The expression patterns of sulfite scavengers including SOX were differentially modulated under cold, heat, salt and high light stresses. Particularly, they tend to be up-regulated under high light and heat while being down-regulated under cold and salt stresses. The presence of cis-regulatory motifs associated with different stresses in upstream regions of SOX genes was thus justified. The protein–protein interaction network of AtSOX and network enrichment with gene ontology (GO) terms showed that most predicted proteins, including sulfite reductase, ATP sulfurylases and APS reductases were among prime enzymes involved in sulfite pathway. Finally, SOX–sulfite docked structures indicated that arginine residues particularly Arg374 is crucial for SOX–sulfite binding and additional two other residues such as Arg51 and Arg103 may be important for SOX–sulfite bindings in plants.     

Essential oil composition of two endemic Centaurea species from Turkey, Centaurea mucronifera and Centaurea chrysantha, collected in the same habitat

The essential oils of two endemic Centaurea species from Turkey, C. mucronifera and C. chrysantha, collected in the same habitat, have been studied. The main compounds of the former were germacrene D (29.3%), β-eudesmol (17.4%) and β-caryophyllene (7.3%), while in the latter germacrene D (27.4%), caryophyllene oxide (9.5%) and bicyclogermacrene (5.4%) were detected among its major constituents. The two species produced many similar compounds in their essential oils that could be justified by the similar ecological conditions of their habitat, but also many differences were found that could confirm their taxonomic separation.     

Trace elements in viral hepatitis

In this study, serum trace elements, including selenium (Se), zinc (Zn), copper (Cu), were determined by using Atomic Absorption Spectrophotometer (SpectrAA 250 Plus Zeeman, Varian, Australia) in sera of patients with viral hepatitis (A, B, C, D, E) cases (n = 102), and statistically compared with the controls (n = 52). In viral hepatitis, Cu levels were found as 3.23 ± 1.02 mg/L, and this value was significantly higher than the control group (1.13 ± 0.21) (p < 0.01). Both, Se and Zn levels found to be significantly low in viral hepatitis cases (p < 0.01). While Se level was 81.4 ± 26.01 μg/L in viral hepatitis (n = 101), it was found to be 166.15 ± 4.58 μg/L in healthy individuals. Meanwhile, Zn levels were 0.230 ± 0.081 mg/L and 0.748 ± 0.392 mg/L in hepatitis cases (n = 101) and the control group, respectively. There was no difference amongst viral hepatitis groups classified in regard with agents and clinical manifestation, such as A, acute hepatitis B, chronic hepatitis B, C, D and E. Previously, it was indicated that absorption disorders in gastrointestinal system, especially in chronic cases, were not main causes of decrease of trace elements by iron and several other parameters in sera of the cases. Therefore, we suggest that decrease in Zn and Se levels and elevation in Cu levels are probably resulted from defence strategies of organism and induced by the hormone-like substances.     

Combined anterolateral thigh flap and vascularized fibula osteoseptocutaneous flap in reconstruction of extensive composite mandibular defects.

Extensive composite defects of the oromandibular area are usually created after the surgical treatment of T3 and T4 cancers, requiring complex reconstructive plastic surgical procedures. The preferred treatment method for this type of defect is reconstruction with two free flaps. The use of the vascularized fibula osteoseptocutaneous flap for the bone and inner lining defect is well known and accepted. Among the flaps that can be used for the outer lining and soft-tissue reconstruction, the two most commonly used have been the forearm flap and the rectus abdominis myocutaneous flap. However, these flaps have some disadvantages that restrict their use for this purpose. The forearm flap is usually too thin to cover the fibular bone and reconstruction plate, and the rectus abdominis myocutaneous flap can cause a subclinical reduction in abdominal strength. Both radial forearm and rectus abdominis myocutaneous flaps are difficult to harvest during tumor excision. Because of these drawbacks, over the past several years the authors have preferred to use the anterolateral thigh flap for outer face, neck, and submandibular region reconstructions. From October of 1998 to June of 2000, 22 extensive composite mandibular defect reconstructions using the free anterolateral thigh flap, combined with the vascularized free fibula osteoseptocutaneous flap, were performed at the Chang Gung Memorial Hospital. Complete flap survival was 90.9 percent (40 of 44 flaps). Complete loss was seen in an anterolateral thigh flap, which was then reconstructed with a pectoralis major myocutaneous pedicled flap (2.3 percent). There were five venous problems: three in osteoseptocutaneous free fibula flaps, the other two in anterolateral thigh flaps; all were revised immediately. However, the skin islands of two osteoseptocutaneous free fibula flaps and one anterolateral thigh flap developed partial necrosis (6.8 percent). The other complications were compartment syndrome in the leg in one patient, external carotid artery rupture in one patient, three donor-site infections in two patients, three neck wound infections, and one myocardial insufficiency; all were treated properly. Thirteen patients underwent revision procedures 6 months after the first operation. These procedures included debulking of the flap or revision of the mouth angle or both. Trismus or intraoral contraction was noted in none of these patients. In conclusion, the free anterolateral thigh flap combined with the vascularized fibula osteoseptocutaneous flap seems to be a good choice in the reconstruction of the extensive composite defects of the oromandibular region aesthetically and functionally.     

Have we found an ideal soft-tissue flap? An experience with 672 anterolateral thigh flaps

The free anterolateral thigh flap is becoming one of the most preferred options for soft-tissue reconstruction. Between June of 1996 and August of 2000, 672 anterolateral thigh flaps were used in 660 patients at Chang Gung Memorial Hospital. Four hundred eighty-four anterolateral thigh flaps were used for head and neck region recontruction in 475 patients, 58 flaps were used for upper extremity reconstruction in 58 patients, 121 flaps were used for lower extremity reconstruction in 119 patients, and nine flaps were used for trunk reconstruction in nine patients. Of the 672 flaps used in total, a majority (439) were musculocutaneous perforator flaps. Sixty-five were septocutaneous vessel flaps. Of these 504 flaps, 350 were fasciocutaneous and 154 were cutaneous flaps. Of the remaining 168 flaps, 95 were musculocutaneous flaps, 63 were chimeric flaps, and the remaining ten were composite musculocutaneous perforator flaps with the tensor fasciae latae. Total flap failure occurred in 12 patients (1.79 percent of the flaps) and partial failure occurred in 17 patients (2.53 percent of the flaps). Of the 12 flaps that failed completely, five were reconstructed with second anterolateral thigh flaps, four with pedicled flaps, one with a free radial forearm flap, one with skin grafting, and one with primary closure. Of the 17 flaps that failed partially, three were reconstructed with anterolateral thigh flaps, one with a free radial forearm flap, five with pedicled flaps, and eight with primary suture, skin grafting, and conservative methods.In this large series, a consistent anatomy of the main pedicle of the anterolateral thigh flap was observed. In cutaneous and fasciocutaneous flaps, the skin vessels (musculocutaneous perforators or septocutaneous vessels) were found and followed until they reached the main pedicle, regardless of the anatomic position. There were only six cases in this series in which no skin vessels were identified during the harvesting of cutaneous or fasciocutaneous anterolateral thigh flaps. In 87.1 percent of the cutaneous or fasciocutaneous flaps, the skin vessels were found to be musculocutaneous perforators; in 12.9 percent, they were found as septocutaneous vessels. The anterolateral thigh flap is a reliable flap that supplies a large area of skin. This flap can be harvested irrespective of whether the skin vessels are septocutaneous or musculocutaneous. It is a versatile soft-tissue flap in which thickness and volume can be adjusted for the extent of the defect, and it can replace most soft-tissue free flaps in most clinical situations.     

Selective oxidation and reduction reactions with cofactor regeneration mediated by galactitol-, lactate-, and formate dehydrogenases immobilized on magnetic nanoparticles

Rapid immobilization with the one-pot purification of galactitol dehydrogenase (GatDH) and formate dehydrogenase (FDH) is achieved by using iminodiacetic acid (IDA) with chelated Co²⁺ modified magnetic nanoparticles as a carrier. Lactate dehydrogenase (LDH) from recombinant Escherichia coli and FDH commencing Candida methylica were used as an auxiliary enzyme for the regeneration of NADH/NAD⁺ with a representative synthesis of (S)-1,2-propanediol and l-tagatose starting from hydroxyacetone and galactitol. The affinity magnetic nanoparticles were characterized by scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR), while the purity of GatDH and FDH was assayed by SDS-PAGE analysis. The immobilized two-enzyme system, reflecting the pH dependence of its constituent enzymes, showed optimal activity at pH 7 and 8 for (S)-1,2-propanediol and l-tagatose production, respectively. The immobilized enzyme system retained up to 70% of its activity after one week of repeated use. The use of affinity magnetic nanoparticles offers the advantage of a one-pot purification of His(6)-tagged GatDH and FDH followed by the production of rare sugar and chiral diol.     

Oral self-nanoemulsifying formulation of GLP-1 agonist peptide exendin-4: development,characterization and permeability assesment on Caco-2 cell monolayer

Amino Acids - The objective of this study was to prepare a stable self-nanoemulsifying formulation of exendin-4, which is an antidiabetic peptide. As exendin-4 is commercially available only in...