Exogenous ascorbic acid (vitamin C) increases resistance to salt stress and reduces lipid peroxidation

The transition from reversible to permanent wilting, in whole tomato seedlings (Lycopersicon esculentum Mill. cv. M82) following severe salt-stress by root exposure to 300 mM NaCl, was investigated. Salinized seedlings wilted rapidly but recovered if returned to non-saline nutrient solution within 6 h. However, after 9 h of salt-treatment 100% of the seedlings remained wilted and died. Remarkably, the addition of an anti-oxidant (0.5 mM ascorbic acid) to the root medium, prior to and during salt-treatment for 9 h, facilitated the subsequent recovery and long-term survival of c. 50% of the wilted seedlings. Other organic solutes without known anti-oxidant activity were not effective. Salt-stress increased the accumulation in roots, stems and leaves, of lipid peroxidation products produced by interactions with damaging active oxygen species. Additional ascorbic acid partially inhibited this response but did not significantly reduce sodium uptake or plasma membrane leakiness.     

A new method of synthesis of fluorescently labelled oligonucleotides and their application in DNA sequencing.

A new approach to the chemical synthesis of oligodeoxynucleotides bearing reporter functional groups at base residues of 3'-end nucleosides is reported. Applications of the 3'-end fluorescently labelled primers for automated DNA sequencing are shown.     

A new benzoic acid derivative from Piper diospyrifolium and its anti-Mycobacterium tuberculosis activity

The extraction by supercritical fluid (SFE-CO₂) from leaves of Piper diospyrifolium and chromatographic column purification afforded the isolation of a new benzoic acid derivative 4-methoxy-3-[(E)-3-methyl-1,3-butadien-1-yl]-5-(3-methyl-2-buten-1-yl)-benzoic acid (1). The chemical structure was elucidated on the basis of spectroscopic methods and comparison with literature data. SFE-CO₂ extracts and (1) were tested for their anti-Mycobacterium tuberculosis activities and cytotoxicities in J774G.8 macrophages. The compound (1) and SFE-CO₂ extracts exhibited moderate activities against M. tuberculosis H₃₇Rv with minimum inhibitory concentration (MIC) values of 125 μg/mL. The MIC values of M. tuberculosis clinical isolates ranged from 125 μg/mL to >250 μg/mL. The cytotoxicities results showed a selectivity index range from 0.6 to 1.0. Additional studies in structure activity-relationship as well as synergistic activity with antituberculous drugs should be conducted for a better evaluation of anti-mycobacterial activity of this compound.     

Radioimmunoassay for a new phenothiazine derivative and its application.

Fluphenazine-4-chlorophenoxy-isobutyrate ester, a new phenothiazine derivative was synthesized in the Institute for Drug Research Budapest. Radioimmunoassay was developed for the therapeutic monitoring of the drug level after intramuscular depot injection. The fluphenazine hapten was coupled to BSA by mixed-anhydride method. Antisera were produced to this conjugation in New-Zealand white rabbits and were tested for the antibody-titer. The specificity was tested by the cross-reaction with phenothiazine-analogues and other psychotropics. Strong cross-reaction was found with compounds possessing piperazine in side chain (trifluoperazine, perphenazine), but other psychotropic drugs did not react. Tritium-labelled trifluoperazine (spec. activity: 3.5 TBq/mmol) was used as a tracer in the radioimmunoassay. The detection limit was 75 pg with a CV of < 5% in 50 microliters plasma sample (equivalent to 1.5 ng/ml concentration) and a standard curve in the 3 ng/ml-50 ng/ml GYKI-22441 concentration range showed a CV of < 10%. Preliminary pharmacokinetic study was performed in Beagle dogs after intramuscular depot injection with GYKI-22441 in sesame oil in a dose of 0.1 mg/kg. The GYKI-22441 concentration of the plasma samples were measured by the RIA method during a 28-day interval after the treatment and was evaluated by the MultiCalc Immunoassay Data Management program (Pharmacia).     

Lipase-catalyzed esterification of unusual substrates: Synthesis of glucuronic acid and ascorbic acid (vitamin C) esters

The synthesis of n-butanol and cinnamic alcohol esters of glucuronic acid and the esterification of ascorbic acid (vitamin C) with phenylbutyric acid was performed with lipase from Candida antarctica B (CAL-B, SP435) in a mainly solid-phase system. Products were obtained in 15 to 22 % yield. Computer modelling based on the structure of CAL-B was used to elucidate the access of glucuronic acid to the catalytic site of the lipase. A fixation of glucuronic acid via H-bonds to Q157, D134 and H224 during the transition state was observed. © Rapid Science Ltd. 1998     

A new short synthesis of 10R-tuberculostearic acid and its enantiomer

Tuberculostearic acid (10R-methyloctadecanoic acid) and its 10S-enantiomer were synthesised by a chiral pool strategy, in four steps from citronellyl bromide.     

A new synthesis of 9-beta-D-ribofuranosyluric acid and its 5'-monophosphate

Syntheses for 9-beta-D-ribofuranosyluric acid (16) and its 5'-monophosphate (14) starting from guanosine and by applying the p-nitrophenylethyl blocking group are described.     

Type III collagen is essential for growth acceleration of human osteoblastic cells by ascorbic acid 2-phosphate, a long-acting vitamin C derivative.

Collagen has been reported to be essential for the proliferation of various kinds of cells including human osteoblastic cells [Takamizawa, S., Maehata, Y., Imai, K., Senoo, H., Sato, S., Hata, R., 2004. Effects of ascorbic acid and ascorbic acid 2-phosphate, a long-acting vitamin C derivative, on the proliferation and differentiation of human osteoblast-like cells. Cell Biol. Int. 28, 255-265], but the type(s) of collagen responsible for growth regulation is not known. Presently we found that ascorbic acid 2-phosphate, a long-acting vitamin C derivative, stimulated both cell growth and the expression of mRNA for type III collagen in human osteoblast-like MG-63 cells and in normal human osteoblasts, as well as in human bone marrow mesenchymal stem cells, but not the expression of type I collagen in these cells. Epidermal growth factor also stimulated both cell growth and expression of type III collagen mRNA in MG-63 cells. Among MG-63 cell clones, their growth rates correlated significantly with their COL3A1 messenger RNA levels but not with their COL1A1 or COL1A2 messenger RNA levels. Transfection of MG-63 cells with siRNA for COL3A1 but not with that for COL1A1 decreased the growth rates of the transfected cells concomitant with a drop in the level of COL3A1 mRNA. Furthermore, cell proliferation as observed by thymidine incorporation into DNA and cell number was increased when MG-63 cells were cultured on type III collagen-coated dishes. Taken together, our results indicate that type III collagen is the collagen component responsible for the growth stimulation of human osteoblastic cells.     

Enzymatic synthesis of alpha-butylglucoside lactate: a new alpha-hydroxy acid derivative

An alpha-hydroxy acid derivative, alpha-butylglucoside lactate, was successfully prepared by enzymatic transesterification of alpha-butylglucoside with a lactate alkyl ester in a non-aqueous medium using immobilized lipase as biocatalyst. Ester synthesis in organic solvent was optimized. Solvent choice was made on the basis of substrate solubility and enzyme stability in the medium. A solvent-free reaction using butyllactate as lactate donor led to the highest yields. In the presence of 0.5M alphabutylglucoside and 100 g/L Novozym(R), a 67 % yield could be obtained within 40 h at 50 degrees C. However, the presence of butanol by-product limited the reaction to a maximum that could not be exceeded in closed systems. The elimination of the alcohol under reduced pressure resulted in the complete equilibrium shift of the transesterification reaction in favor of synthesis; below 15 mbars, more than 95% of 0.5M alpha-butylglucoside could be converted within 30 h. Moreover, simultaneous evaporation of water allowed hydrolysis of butyllactate to be eliminated. Consequently, a very high alpha-butylglucoside lactate concentration (170 g/) could be obtained in a single batch reaction. A single purification procedure, consisting of butyllactate extraction with hexane, enabled the product to be obtained at a purity above 95% (w/w). 1H and 13C NMR analysis later demonstrated that lactic acid was exclusively grafted onto the primary hydroxyl group of alphabutylglucoside.     

Progressive pseudogenization: vitamin C synthesis and its loss in bats

For the past 50 years, it was believed that all bats, like humans and guinea pigs, did not synthesize vitamin C (Vc) because they lacked activity of L-gulonolactone oxidase (GULO) in their livers. Humans and guinea pigs lack the activity due to pseudogenization of GULO in their genomes, but there is no genetic evidence to show whether such loss in bats is caused by pseudogenization. Unexpectedly, our successful molecular cloning in one frugivorous bat (Rousettus leschenaultii) and one insectivorous bat (Hipposideros armiger) ascertains that no pseudogenization occurs in these species. Furthermore, we find normal GULO protein expression using bat-specific anti-GULO polyclonal antibodies in bats, evaluated by Western blotting. Most surprisingly, GULO activity assays reveal that these two bat species have retained the ability to synthesize Vc, but at low levels compared with the mouse. It is known that bats in the genus Pteropus have lost GULO activity. We then found that functional constraints acting on the GULO of Pteropus vampyrus (which lost its function) are relaxed. These results imply that the ability to synthesize Vc in bats has not been lost completely in species as previously thought. We also suggest that the evolution of bat GULO genes can be a good model to study genetic processes associated with loss-of-function.     

A new synthesis of cerebrosterol and its 24-epimer from lithocholic acid

A new method for the synthesis of both isomers of 24-hydroxycholesterol starting from lithocholic acid is proposed.     

A new synthesis of cerebrosterol and its 24-epimer from lithocholic acid

A new method for the synthesis of both isomers of 24-hydroxycholesterol starting from lithocholic acid is proposed.     

A convenient synthesis of labelled rhodopsin and studies on its active site

Digitonin solutions of labelled rhodopsin, containing (3)H in the retinyl moiety, were prepared by two related methods. Labelled rhodopsin was also prepared for the first time in cetyltrimethylammonium bromide and purified by column chromatography. It was shown that only certain rhodopsin preparations on denaturation in the dark and the reduction with sodium borohydride gave up to 60% of the radioactivity in a fraction characterized as N-retinylphosphatidylethanolamine. Such preparations also gave a lipid-linked retinyl moiety at the metarhodopsin-I stage, but, as expected, a protein-linked retinyl moiety at the metarhodopsin-II stage. Other preparations however, gave exclusively protein-bound radioactivity at the native-rhodopsin, metarhodopsin-I and metarhodopsin-II stages. It is therefore conceivable that the formation of N-retinylphosphatidylethanolamine is due to a non-enzymic reaction resulting from the transfer of the retinyl moiety from its native site to an amino group of a favourably oriented phospholipid molecule. The only firmly established aspect of the rhodopsin active site remains the demonstration in our previous work that at the metarhodopsin-II stage the retinyl moiety is linked to an in-amino group of lysine. On the basis of chemical reactivity it is argued that the light-induced conversion of rhodopsin into metarhodopsin II involves a profound conformational change resulting in the dislocation of the retinylideneiminium chromophore from a non-polar environment in rhodopsin to a polar environment in metarhodopsin II.