The cytomegalovirus enhancer: a pan-active control element in transgenic mice.

In an effort to identify widely active positive regulatory elements, we have examined the action of the cytomegalovirus enhancer-promoter in transgenic mice. These elements activated expression in 24 of 28 tissues tested. The greatest expression was observed in the heart, kidney, brain, and testis. Maximum expression further localized to specific cells within the heart and kidney.     

Biotransformation of 2,2-Dimethyl-1,3-Propanediol to 3-Hydroxypivalic Acid by Acetobacter Acetii DSMZ3508 and Related Bacteria

Acetobacter acetii DSMZ3508 and related bacteria converted 2,2-dimethyl-1,3-propanediol into 3-hydroxypivalic acid (2,2-dimethyl-3-hydroxypropionic acid; 3HP) during submerged cultivation in mineral salt medium. The maximum yield of 3-hydroxypivalic acid was 24.4% of the fed substrate after 18 days. Cultivation parameters, as pH, cell density, optimal substrate concentration, and oxygen supply for the bioconversion process were determined.     

Regulation of thyroid hormone metabolism in rat liver fractions

The nature of the conversion of thyroxine (T₄) to triiodothyronine (T₃) and reverse triiodothyronine (rT₃) was investigated in rat liver homogenate and microsomes. A 6-fold rise of T₃ and 2.5-fold rise of rT₃ levels determined by specific radioimmunoassays was observed over 6 h after the addition of T₄. An enzymic process is suggested that converts T₄ to T₃ and rT₃. For T₃ the optimal pH is 6 and for rT₃, 9.5. The converting activity for both T₃ and rT₃ is temperature dependent and can be suppressed by heat, H₂O₂, merthiolate and by 5-propyl-2-thiouracil. rT₃ and to a lesser degree iodide, were able to inhibit the production of T₃ in a dose related fashion. Therefore the pH dependendy, rT₃ and iodide may regulate the availability of T₃ or rT₃ depending on the metabolic requirements of thyroid hormones.     

Creatine Transport in Cultured Cells of Rat and Mouse Brain

Astroglia-rich cultures derived from brains of newborn rats or mice use a transport system for the uptake of creatine. The uptake system is saturable, Na+-dependent, and highly specific for creatine and Na+. Kinetic studies on rat cells revealed a Km value for creatine of 45 microM, a Vmax of 17 nmol x h-1 x (mg of protein)-1, and a Km value of 55 mM for Na+. The carrier is competitively inhibited by guanidinopropionate (Ki = 15 microM). No such transport system was found in neuron-rich primary cultures from embryonic rat brain. It is hypothesized that creatine transport is an astroglial rather than a neuronal function.     

The gap junctional intercellular communication is no prerequisite for the stabilization of xenobiotic metabolizing enzyme activities in primary rat liver parenchymal cells in vitro

Summary In primary monocultures of adult rat liver parenchymal cells (PC), the activities of the xenobiotic metabolizing enzymes microsomal epoxide hydrolase (mEH_b), soluble epoxide hydrolase (sEH), glutathione S-transferases (GST), and phenolsulfotransferase (ST) were reduced after 7 d to values below 33% of the initial activities. Furthermore, the gap junctional intercellular communication (GJIC), measured after microinjection by dye transfer, decreased from 90% on Day 1 to undetectable values after 5 d in monoculture. Co-culture of PC with nonparenchymal rat liver epithelial cells (NEC) increased (98% on Day 1) and stabilized (82% on Day 7) the homotypic GJIC of PC. Additionally, most of the measured xenobiotic metabolizing enzyme activities were well stabilized over 1 wk in co-culture. Because GJIC is one of several mechanisms playing an important role in cell differentiation, the importance of GJIC for the stabilization of xenobiotic metabolizing enzymes in PC was investigated. PC in monoculture were, therefore, treated with 2% dimethyl sulfoxide (DMSO), a differentiation promoting factor, and 1,1,1-trichloro-2,2,-bis (p-chlorophenyl) ethane (DDT) (10 μg/ml), a liver tumor promotor and inhibitor of GJIC, was given to co-cultures of PC with NEC. DMSO significantly stabilized (68% on Day 7), while DDT significantly inhibited (8% on Day 7) homotypic GJIC of PC in the respective culture systems. In contrast, the activities of mEH_b, sEH, GST, and ST were not affected in the presence of DMSO or DDT. These results lead to the assumption that the differentiation parameters measured in this study (i.e., homotypic GJIC and the activities of xenobiotic metabolizing enzymes) are independently regulated in adult rat liver PC.     

Cloning and expression analysis of an ADP-ribosylation factor from Solanum tuberosum L.

Summary A cDNA clone encoding an ADP-ribosylation factor from potato (Solanum tuberosum L.) was isolated. The nucleotide and deduced amino acid sequences show high homology to known ADP-ribosylation factor sequences from Arabidopsis, yeast, cow and man. In northern blot experiments, all tissues analysed showed expression of the corresponding mRNA. Strongest expression was found, however, in potato tubers.Abbreviations ARF, ADP ribosylation factor - BSA bovine serum albumin - EDTA ethylenediaminetetraacetic acid - SDS sodium dodecyl sulfate - SSC sodium saline citrate     

ABH antigens as recognition sites for the activation of red blood cell anion exchange by the lectin Ulex europaeus agglutinin I

The blood group antigen H (blood group O) and fucose-specific lectin Ulex europaeus agglutinin I (UEA₁) (10 μg/ml) was found to increase the rate constant of CL^? efflux into 100mM Na⁺ oxalate media by about 40% in erythrocytes taken from antigen H donors. In 100 mMK ⁺ oxalate, 150 mM Na⁺ pyruvate and in 150 mM Na⁺ acetate media the lectin elevated the rate constant of CL^? efflux by 20–50%. The acceleration of Cl^? efflux by UEA₁ was completely blocked by 10 μM 4,4′-dllsothiocyanato-stilbene-2,2′-disulfonic acid (DIDS) indicating that the effect of the lectin is mediated by the anion exchanger of human erythrocytes (band 3 protein). In antigen A₁ erythrocytes no significant stimulation of anion exchange by UEA₁ was seen. The activation of Cl^? efflux was completely prevented by addition of 1 mM fucose to the medium. These results suggest that the effect of UEA₁ is mediated through interaction with the fucose residues of H antigens. Increasing extracellular Ca⁺⁺ from 0.5 to 5 mM in Na ⁺ pyruvate or Na⁺ acetate media slightly reduced the acceleration of anion exchange by the lectin. On the other hand, replacing part of extracellular chloride by bicarbonate did not considerably alter the (previously reported) stimulatory effect of UEA₁ on red blood cell Ca⁺⁺ uptake. This suggests that the acceleration of anion exchange and of Ca⁺⁺ uptake by UEA₁, respectively, are mediated by different mechanisms. It is concluded that UEA₁ activates anion exchange of human erythrocytes most probably by a direct interaction with H antigens present on extracellular domains of the band 3 protein. © 1993 Wiley-Liss, Inc.     

Reaction intensification for biocatalytic production of polyphenolic natural product di-C-β-glucosides

Polyphenolic aglycones featuring two sugars individually attached via C-glycosidic linkage (di-C-glycosides) represent a rare class of plant natural products with unique physicochemical properties and biological activities. Natural scarcity of such di-C-glycosides limits their use-inspired exploration as pharmaceutical ingredients. Here, we show a biocatalytic process technology for reaction-intensified production of the di-C-β-glucosides of two representative phenol substrates, phloretin (a natural flavonoid) and phenyl-trihydroxyacetophenone (a phenolic synthon for synthesis), from sucrose. The synthesis proceeds via an iterative two-fold C-glycosylation of the respective aglycone, supplied as inclusion complex with 2-hydroxypropyl β-cyclodextrin for enhanced water solubility of up to 50 mmol/L, catalyzed by a kumquat di-C-glycosyltransferase (di-CGT), and it uses UDP-Glc provided in situ from sucrose by a soybean sucrose synthase, with catalytic amounts (≤3 mol%) of UDP added. Time course analysis reveals the second C-glycosylation as rate-limiting (0.4–0.5 mmol/L/min) for the di-C-glucoside production. With internal supply from sucrose keeping the UDP-Glc at a constant steady-state concentration (≥50% of the UDP added) during the reaction, the di-C-glycosylation is driven to completion (≥95% yield). Contrary to the mono-C-glucoside intermediate which is stable, the di-C-glucoside requires the addition of reducing agent (10 mmol/L 2-mercaptoethanol) to prevent its decomposition during the synthesis. Both di-C-glucosides are isolated from the reaction mixtures in excellent purity (≥95%), and their expected structures are confirmed by NMR. Collectively, this study demonstrates efficient glycosyltransferase cascade reaction for flexible use in natural product di-C-β-glucoside synthesis from expedient substrates.     

Biosynthesis and action of nitric oxide in mammalian cells

Nitric oxide (NO) can act as a vasorelaxant, a modulator of neurotransmission and a defence against pathogens. However, under certain conditions, NO can also have damaging effects to cells. Whether NO is useful or harmful depends on its chemical fate, and on the rate and location of its production. Here, we discuss progress in NO chemistry and the enzymology of NO synthases, and we will also attempt to explai its actions in the cardiovascular, nervous and immune systems.