A bioprocess for the production of high concentrations of R-(+)-α-terpineol from R-(+)-limonene

A bioprocess with a high conversion rate of limonene to α-terpineol was described. The enzyme hydratase involved in this process was found as being cofactor independent, non-inducible and able to perform the transformation of both R-(+) and S-(−)-limonene. The system used consisted of a biphasic medium in which the aqueous phase contained a concentrated resting cells of Sphingobium sp. and the organic phase was sunflower oil. After 30 h at 30 °C ca. 25 g of R-(+)-α-terpineol per liter of organic phase were obtained from R-(+)-limonene in Erlenmeyer flasks. Performance of the bioconversion in a bioreactor increased the production rate with no changes in yield and maximal R-(+)-α-terpineol concentration, which demonstrated that experiments in flasks were limited by liquid–liquid transport phenomena. A mathematical model able to explain the fact that the reaction always stopped before the precursor became exhausted has also been proposed and validated. Finally, the process reported was the most promising alternative for the biotechnological production of natural R-(+)-α-terpineol published so far and up to ca. 130 g L⁻¹ metabolite could finally be obtained.     

Role of the frequency of blood CD4(+) CXCR5(+) CCR6(+) T cells in autoimmunity in patients with Sjögren's syndrome

The blood CD4⁺ CXCR5⁺ T cells, known as “circulating” Tfh, have been shown to efficiently induce naïve B cells to produce immunoglobulin. They play an important role in certain autoimmune diseases. In the present study, we show for the first time that the frequency of CD4⁺ CXCR5⁺ T cells is increased in pSS patients and positively correlated with autoantibodies in the blood. The concentration of Th17-like subsets (CD4⁺ CXCR5⁺ CCR6⁺) in pSS patients was found to be significantly higher than in healthy controls. Functional assays showed that activated Th17-like subtypes in the blood display the key features of Tfh cells, including invariably coexpressed PD-1, ICOS, CD40L and IL-21. Th17 subsets were found to highly express Bcl-6 protein and Th1 and Th2 were not. Bcl-6 is believed to be a master transforming factor for Tfh cell differentiation and facilitate B cell proliferation and somatic hypermutation within the germinal center. These data indicate that Th17 subsets of CD4⁺ CXCR5⁺ T cells in the blood may participate in the antibody-related immune responses and that high frequency of CD4⁺ CXCR5⁺ CCR6⁺ Tfh cells in blood may be suitable biomarkers for the evaluation of the active immune stage of pSS patients. It might provide insights into the pathogenesis and perhaps help researchers identify novel therapeutic targets for pSS.     

SA-4-1BBL Costimulation Inhibits Conversion of Conventional CD4(+) T Cells into CD4(+)FoxP3(+) T Regulatory Cells by Production of IFN-γ

Tumors convert conventional CD4⁺ T cells into induced CD4⁺CD25⁺FoxP3⁺ T regulatory (iTreg) cells that serve as an effective means of immune evasion. Therefore, the blockade of conventional CD4⁺ T cell conversion into iTreg cells represents an attractive target for improving the efficacy of various immunotherapeutic approaches. Using a novel form of 4-1BBL molecule, SA-4-1BBL, we previously demonstrated that costimulation via 4-1BB receptor renders both CD4⁺and CD8⁺ T effector (Teff) cells refractory to inhibition by Treg cells and increased intratumoral Teff/Treg cell ratio that correlated with therapeutic efficacy in various preclinical tumor models. Building on these studies, we herein show for the first time, to our knowledge, that signaling through 4-1BB inhibits antigen- and TGF-β-driven conversion of naïve CD4⁺FoxP3⁻ T cells into iTreg cells via stimulation of IFN-γ production by CD4⁺FoxP3⁻ T cells. Importantly, treatment with SA-4-1BBL blocked the conversion of CD4⁺FoxP3⁻ T cells into Treg cells by EG.7 tumors. Taken together with our previous studies, these results show that 4-1BB signaling negatively modulate Treg cells by two distinct mechanisms: i) inhibiting the conversion of CD4⁺FoxP3⁻ T cells into iTreg cells and ii) endowing Teff cells refractory to inhibition by Treg cells. Given the dominant role of Treg cells in tumor immune evasion mechanisms, 4-1BB signaling represents an attractive target for favorably tipping the Teff:Treg balance toward Teff cells with important implications for cancer immunotherapy.     

Modulation of Na(+)-K(+)-ATPase cell surface abundance through structural determinants on the α1-subunit

Through their ion-pumping and non-ion-pumping functions, Na(+)-K(+)-ATPase protein complexes at the plasma membrane are critical to intracellular homeostasis and to the physiological and pharmacological actions of cardiotonic steroids. Alteration of the abundance of Na(+)-K(+)-ATPase units at the cell surface is one of the mechanisms for Na(+)-K(+)-ATPase regulation in health and diseases that has been closely examined over the past few decades. We here summarize these findings, with emphasis on studies that explicitly tested the involvement of defined regions or residues on the Na(+)-K(+)-ATPase α1 polypeptide. We also report new findings on the effect of manipulating Na(+)-K(+)-ATPase membrane abundance by targeting one of these defined regions: a dileucine motif of the form [D/E]XXXL[L/I]. In this study, opossum kidney cells stably expressing rat α1 Na(+)-K(+)-ATPase or a mutant where the motif was disrupted (α1-L499V) were exposed to 30 min of substrate/coverslip-induced-ischemia followed by reperfusion (I-R). Biotinylation studies suggested that I-R itself acted as an inducer of Na(+)-K(+)-ATPase internalization and that surface expression of the mutant was higher than the native Na(+)-K(+)-ATPase before and after ischemia. Annexin V/propidium iodide staining and lactate dehydrogenase release suggested that I-R injury was reduced in α1-L499V-expressing cells compared with α1-expressing cells. Hence, modulation of Na(+)-K(+)-ATPase cell surface abundance through structural determinants on the α-subunit is an important mechanism of regulation of cellular Na(+)-K(+)-ATPase in various physiological and pathophysiological conditions, with a significant impact on cell survival in face of an ischemic stress.     

Potential α-glucosidase inhibitors from thermal transformation of (+)-catechin

Thermal transformation of the (+)-catechin (1) with heating processing afforded a new oxidation product, gambiriin D (2), along with catechin [6′–8]-catechin (3), and (+)-epicatechin (4). The structure of a new catechin dimer with C–C linkage was determined on the basis of spectroscopic data interpretation. The catechin dimers 2 and 3 exhibited significantly improved inhibitory activities against α-glucosidase, with IC₅₀ values of 0.16 ± 0.2 and 0.14 ± 0.2 μM, respectively, when compared to parent (+)-catechin. Kinetic analysis showed that the two effective compounds 2 and 3 have noncompetitive modes of action.     

Enzymatic activity of cell-free extracts from Burkholderia oxyphila OX-01 bio-converts (+)-catechin and (−)-epicatechin to (+)-taxifolin

This study characterized the enzymatic ability of a cell-free extract from an acidophilic (+)-catechin degrader Burkholderia oxyphila (OX-01). The crude OX-01 extracts were able to transform (+)-catechin and (?)-epicatechin into (+)-taxifolin via a leucocyanidin intermediate in a two-step oxidation. Enzymatic oxidation at the C-4 position was carried out anaerobically using H₂O as an oxygen donor. The C-4 oxidation occurred only in the presence of the 2R-catechin stereoisomer, with the C-3 stereoisomer not affecting the reaction. These results suggest that the OX-01 may have evolved to target both (+)-catechin and (?)-epicatechin, which are major structural units in plants.     

How regulatory CD25(+)CD4(+) T cells impinge on tumor immunobiology? On the existence of two alternative dynamical classes of tumors

Aiming to get a better insight on the impact of regulatory CD25(+)CD4(+) T cells in tumor immunobiology, a simple mathematical model was formulated and studied. This model is an extension of a previous model for the dynamics of autoreactive regulatory cells and effector cells that interact upon their co-localized activation at the antigen presenting cells (APCs). It assumes that tumor growth stimulates the activation and migration to the adjacent lymph node of fresh APCs loaded with tumor antigens. These APCs stimulate the growth of both effector and regulatory T cells, which may then migrate to the tumor site and induce tumor cell destruction. Our results predict the existence of two alternative dynamic modes of unbounded tumor growth. In the first mode, the tumor induces the expansion of effector T cells that outcompete regulatory T cells, but nevertheless fail to control the tumor. In the second mode, the tumor induces a balanced expansion of both effector and regulatory T cells, which prevents the tumor from being destroyed by the immune cells. Tumors characterized by a high specific growth rate, low immunogenicity, and that are relatively resistant to T cell destructive functions, will grow in the first mode; conversely, tumors that have a slow specific growth rate, that are immunogenic, and/or that are more sensitive to destruction by T cells will grow in the second mode. Overall, this result provides a simple explanation to the fact that the development of some tumors expands regulatory T cells while others do not, predicting how some key dynamical properties of the tumor determine either one or the other type of behavior.     

Expansion of CD14(+)CD16(+) monocytes producing TNF-α in complication-free diabetes type 1 juvenile onset patients

We concentrated on the complication-free phase of juvenile onset type 1 diabetes mellitus (T1DM) searching for associations between concentration of inflammatory factors TNF-α, CRP and VEGF and two monocyte subsets the CD14(++)CD16(-) and CD14(+)CD16(+). We analysed a randomly selected group of 150 patients without complications (disease duration 2.74±2.51years) at the start of the project and 5years later. They were compared with 24 patients with retinopathy (6.53±3.39years of disease) and 30 healthy volunteers. Our results indicate that in the complication-free period the concentration of TNF-α significantly increased and continued to increase after retinopathy was established. After 5years the percentage and absolute number of CD14(+)CD16(+) monocytes doubled in complication-free patients. Our study indicates that the size of CD14(+)CD16(+) monocyte subset may be used alternatively to CRP values as an indicator of inflammation grade. Our results imply the necessity of trials using anti-TNF-α therapy in the complication-free phase of the disease.     

Biotransformation of (−)-epicatechin, (+)-epicatechin, (−)-catechin,and (+)-catechin by intestinal bacteria involved in isoflavone metabolism

Isoflavone-metabolizing bacteria, Adlercreutzia equolifaciens, Asaccharobacter celatus, Slackia equolifaciens, and Slackia isoflavoniconvertens catalyzed C-ring cleavage of (–)-epicatechin and (+)-catechin, (+)-epicatechin, and (–)-catechin in varying degrees. The cleaving abilities of (–)-epicatechin and (+)-catechin were enhanced by hydrogen, except (+)-catechin cleavage by S. equolifaciens, which was not accelerated. (?)-Catechin cleavage by Ad. equolifaciens was remarkably accelerated by hydrogen.     

A 133Cs nuclear magnetic resonance study of endothelial Na(+)-K(+)-ATPase activity: can actin regulate its activity?

Using (133)Cs+ NMR, we developed a technique to repetitively measure, in vivo, Na(+)-K(+)-ATPase activity in endothelial cells. The measurements were made without the use of an exogenous shift reagent, because of the large chemical shift of 1.36 +/- 0.13 ppm between intra- and extracellular Cs+. Intracellularly we obtained a spin lattice relaxation time (T1) of 2.0 +/- 0.3 s, and extracellular T1 was 7.9 +/- 0.4 s. Na(+)-K+ pump activity in endothelial cells was determined at 12 +/- 3 nmol Cs+ x min(-1) x (mg Prot)[-1] under control conditions. When intracellular ATP was depleted by the addition of 5 mM 2-deoxy-D-glucose (DOG) and NaCN to about 5% of control, the pump rate decreased by 33%. After 80 min of perfusion with 5 mM DOG and NaCN, reperfusion with control medium rapidly reestablished the endothelial membrane Cs+ gradient. Using (133)Cs+ NMR as a convenient tool, we further addressed the proposed role of actin as a regulator of Na(+)-K+ pump activity in intact cells. Two models of actin rearrangement were tested. DOG caused a rearrangement of F-actin and an increase in G-actin, with a simultaneous decrease in ATP concentration. Cytochalasin D, however, caused an F-actin rearrangement different from that observed for DOG and an increase in G-actin, and cellular ATP levels remained unchanged. In both models, the Na(+)-K(+)-pump activity remained unchanged, as measured with (133)Cs NMR. Our results demonstrate that (133)Cs NMR can be used to repetitively measure Na(+)-K(+)-ATPase activity in endothelial cells. No evidence for a regulatory role of actin on Na(+)-K(+)-ATPase was found.     

HBeAg(+)或抗-Hbe(+)病人血清中HBV标志检测临床意义

近年来,由于乙肝病毒(HBV)分子生物学研究取得很大进展,为HBV标志物检测提供了灵敏的方法,从而使临床评价HBV标志物的工作得到广泛开展[1]。本文报告90份HBeAg( )和56份抗HBe( )病人血清HBV标志物检测结果,探讨其临床意义。临床资料1检测血清本文检测血清标本均为我院住院病人,HBeAg( )病人90例,其中急性乙型肝炎(急肝)13例,慢性活动性肝炎(慢迁肝)39例,慢性迁延性肝炎(慢迁肝)31例,肝硬化7例。抗Hbe( )病人56例,其中急肝5例,慢活肝13例,慢迁肝29例,肝硬化9例。肝炎诊断依据1990年第六次全国肝炎会议修订标准,肝硬化诊断依据内科学…     

Changes of γ-linolenic acid content inRhizopus arrhizus duringl(+)-lactic acid fermentation

Effects of nitrogen source, temperature and pH onl(+)-lactic acid production and γ-linolenic acid (GLA) accumulation byRhizopus arrhizus were examined. The nitrogen source had a minor effect on lactate synthesis but influenced the total lipid content and the fatty acid composition in fungus. Higher temperature favorably influenced the rate of both lactic acid production and lipid formation in the biomass and caused a decrease in the yields of oligounsaturated fatty acids. At higher temperature and after glucose exhaustion, degradation of lactate increased. A low pH value negatively affected the formation of lipids and lactate synthesis. The highest value of GLA in the lipid (25.5%,W/W) was reached at the end of lactate synthesis, but maximum yields of total lipids were achieved when the cultivation continued in the presence of lactate until polyols were exhausted.     

Microbial transformations of (−)-α- and (+)-β-thujone by fungi of Absidia species

The microbial transformations of (−)-α- and (+)-β-thujone (1a and 1b) in cultures of Absidia species: Absidia coerulea AM93, Absidia glauca AM254 and Absidia cylindrospora AM336 were studied. The biotransformations of (−)-α-thujone (1a), by these fungi strains, afforded mixtures of 4-hydroxy- and 7-hydroxy-α-thujone (2 and 3). Aforementioned fungi strains were also able to hydroxylate of (+)-β-thujone at C-7 position. Only A. glauca AM254 transformed 1b to 8-hydroxy-β-thujone (7) and (2S)-2-hydroxyneoisothujol (6). The (4R)-4-hydroxyisothujole (5) was identified as one of the major metabolite of (+)-β-thujone (1b) in culture of A. cylindrospora AM336. This strain was also able to introduce hydroxy group to C-4 position in 1b without reduction of carbonyl group at C-3. The absolute configuration of all chiral centers of new (4R)-4-hydroxyisothujol (5) and (2S)-2-hydroxyneoisothujol (6) were established taking into account the configuration of (+)-β-thujone (1b) and their spectral data.     

Different responses of CR(−) and CR(+) B cells to LPS stimulation

Proliferative response of B cells with or without CR [CR(+) or CR(?) B cells] was compared in their polyclonal response when they were stimulated with lipopolysaccharide (LPS). CR(+) B cells responded better in proliferation and more poorly in polyclonal antibody formation than did CR(?) B cells. The dissociation between proliferation and antibody formation in LPS response was not due to the shift of the time kinetics nor the exhaustion of the culture medium. T cells and macrophages did not take part in the dissociation, since macrophage depletion from nu/nu mouse spleen cells could not modify the dissociation. The polyclonal antibody response of CR(?) B cells was more resistant to irradiation than that of CR(+) B cells. These results suggest that among LPS-responsive B cells there are CR(?) B-cell subset(s) more mature than CR(+) B cells.     

Semi-preparative chromatographic purification of the enantiomers S-(−)-amlodipine and R-(+)-amlodipine

Pharmacokinetic studies of optically pure compounds after single enantiomer administration are becoming increasingly important. The process of racemization in vivo can diminish all expected advantages of single enantiomer treatment. Amlodipine, one of the calcium channel blockers, currently used in therapy as a racemate, is one of such drugs under study. In order to administer single enantiomers of amlodipine to healthy volunteers both were chromatographically purified and characterised. The two optical isomers of amlodipine, active S-(−)- and non-active R-(+)-amlodipine, were purified using chromatographic procedure adopted from the analytical separation. Enantiomers were successfully converted to benzenesulphonic salt without any racemization. All semi-preparative purifications were monitored with complementary analytical methods, HPLC and CE, along with the determination of optical activity so that the final product was sufficiently defined for further in vivo studies. The analytical method developed for the determination of plasma concentrations of each enantiomer of amlodipine in these studies is also briefly described.     

Proteomic analysis of CD44(+) and CD44(−) gastric cancer cells

CD44 is a cell surface protein and it is widely used as a cancer stem cell marker in various cancer types including gastric cancer. We conducted proteomic analysis in CD44(+) and CD44(?) gastric cancer cells to understand characteristics of CD44(+) and CD44(?) cells. In the present study, we sorted cells from the gastric cancer cell line MKN45 according to CD44 expression to separate out CD44(+) and CD44(?) cells. And we conducted RT-PCR to identify mRNA expression of cancer stem cell markers in CD44(+) and CD44(?) cells. Cancer stem cell markers showed upregulated expression in CD44(+) cells. Next, we performed two-dimensional electrophoresis analysis to determine the differential expression pattern of proteins in each group; control, CD44(+), and CD44(?) MKN45 cells. We found a total of 113 spots that varied in expression between CD44(+) and CD44(?) cells, and subjected 20 of those protein spots to MALDI-MS. We selected the three proteins (HSPA8; heat shock cognate 71 kDa protein isoform 1, ezrin, α-enolase) upregulated in CD44(+) cells than CD44(?) cells and one protein (prohibitin) showed increased expression in CD44(?) cells. We validated the protein expression levels of four selected proteins by Western blot. We suggest that our study could be a helpful background to study CD44(+) cancer stem-like cells and differences between CD44(+) and CD44(?) cells in gastric cancer.     

Automated Measurement of d(−), l(+)-Lactate in Soy Sauce and Wine

Rapid, convenient and accurate quantitative assay method of d (?) and l (+)-lactate in soy sauce or wine was established by the combination of a new carboxylic acid analyzer and a discrete type auto analyzer with the aid of l (+)-Iactate dehydrogenase. This method does not require specific pretreatment of any samples and samples can be analyzed only after the dilution, filtration or deproteinization.     

Does the KdpA subunit from the high affinity K(+)-translocating P-type KDP-ATPase have a structure similar to that of K(+) channels?

Evidence is presented that the transmembrane KdpA subunit of the high affinity K(+)-translocating P-type Kdp-ATPase is evolutionarily derived from the superfamily of 2TM-type K(+) channels in bacteria. This extends a previous study relating the K(+) channels to the KtrAB, Trk, Trk1,2, and HKT1 K(+) symporter superfamily of both prokaryotes and eukaryotes. Although the channels are formed by four single-MPM motif subunits, the transmembrane KdpA subunit and the transmembrane subunit of the symporter proteins are postulated to have four corresponding MPM motifs within a single sequence. Analysis of 17 KdpA sequences reveals a pattern of residue conservation similar to that of the symporters and channels, and consistent with the crystal structure of the KcsA K(+) channel. In addition, the most highly conserved residues between the families, specifically the central glycines of the P2 segments, are those previously identified as crucial for the property of K(+)-selectivity that is common to each protein. This hypothesis is consistent with an experimental study of mutations that alter K(+) binding affinity of the Kdp transporter. Although most of the results of a previous study of the transmembrane topology of KdpA are consistent with the 4-MPM model, the one deviation can be explained by a plausible change in the structure due to the experimental method.