Degradation of malic acid in wine by immobilized Issatchenkia orientalis cells with oriental oak charcoal and alginate.

AIMS: To test degradation of malic acid content in wine by immobilized Issatchenkia orientalis KMBL 5774 cells recently isolated from Korean wine pomace as a malic acid-degrading yeast. METHODS AND RESULTS: I. orientalis KMBL 5774 cells were immobilized using a mixture of oriental oak (Quercus variabilis) charcoal with sodium alginate. When the immobilized yeast cells were observed on a scanning electron microscope, cells were efficiently immobilized on the surface area of the charcoal. A Korean wine containing a high level of malic acid was treated with the immobilized yeast cells. The HPLC analysis of the malic acid content in the treated wine showed the malic acid content was reduced to 0.75 mg ml(-1) after treatment from the original content of 8.96 mg ml(-1), representing 91.6% of the malic acid was degraded during the treatment. CONCLUSIONS: The immobilization of the malic acid-degrading yeasts with oriental oak charcoal and sodium alginate is useful for degradation of malic acid in wines containing a high level of malic acid with no significant increase in other acids. SIGNIFICANCE AND IMPACT OF THE STUDY: Malic acid is sometimes detrimental to the quality of wines when present at high concentrations in some varieties. The immobilized I. orientalis KMBL5774 cells appear to be a promising candidate in view of developing biotechnological methods for reduction of malic acid contents in wine.

Bioactive constituents of Leptadenia arborea.

The aerial part of Leptadenia arborea has been shown to contain pinoresinol (1), syringaresinol (2), leucanthemitol (3) and E-ferulaldehyde (4). These known compounds are being reported for the first time from this plant. Among them, syringaresinol has shown an inhibitory effect against acetylcholinesterase. The IC(50) (the concentration of 50% enzyme inhibition) value of this compound was 200 microg/ml.

An improved method for determination of ethyl carbamate in Korean traditional rice wine.

An improved extraction method for ethyl carbamate, a genotoxic and carcinogenic compound found in various fermented foods and beverages, was investigated for its determination in the two most typical Korean traditional rice wines, takju and yakju. When the rice wines were extracted twice with chloroform at 30 degrees C for 60 min, the recovery of ethyl carbamate was less than 16%. When they were saturated with NaCl before extraction, the recovery of ethyl carbamate increased to 24.4% in takju and 67.2% in yakju. Adjustment of pH to 9.0 after NaCl saturation in takju resulted in a dramatic increase of recovery to 81.2%, but not in yakju. When the contents of ethyl carbamate and its precursor, urea, in various Korean traditional rice wines were determined, there was no correlation between the two contents. This is due to the fact that storage time is more important than urea content in the formation of ethyl carbamate in rice wine. In addition, its storage at high temperature resulted in a dramatic increase in ethyl carbamate content according to the prolonged storage time, suggesting that storage time and temperature play a key role in the formation of ethyl carbamate in Korean traditional rice wine.

Purification and characterization of a novel extracellular protease from Bacillus cereus KCTC 3674.

Bacillus cereus KCTC 3674 excretes several kinds of extracellular proteases into the growth medium. Two proteases with molecular masses of approximately 36-kDa and 38-kDa, as shown by SDS-PAGE, were purified from the culture broth. The 38-kDa protease was purified from B. cereus cultivated at 37 degrees C, and the 36-kDa protease was obtained from the B. cereus cultivated at 20 degrees C. The 38-kDa protease was identified as an extracellular neutral (metallo-) protease and was further characterized. The 36-kDa protease was shown to be a novel enzyme based on its N-terminal amino acid sequence, its identification as a metallo-enzyme that was strongly inhibited by EDTA and o-phenanthroline, its hemolysis properties, and its optimal pH and temperature for activity of 8.0 and 70 degrees C, respectively.

Screening for acetylcholinesterase inhibitors from Amaryllidaceae using silica gel thin-layer chromatography in combination with bioactivity staining.

Thin-layer chromatography (TLC) was used to screen for acetylcholinesterase inhibitors from Amaryllidaceae extracts. The TLC plate was developed and then stained using Ellman's reagent, 5,5'-dithiobis-(2-nitrobenzoic acid), to detect acetylcholinesterase activity. The advantages of this TLC assay method were that we could dereplicate the known inhibitor galanthamine, widely occurring in Amaryllidaceae, at an early stage of the isolation procedure. Moreover, there is no disturbance from sample dissolving solvents as in the microplate assay, and it is a very simple method. The detection limits were 10-200 ng for several known acetylcholinesterase inhibitors tested, and it is thus more sensitive than UV or Dragendorff's reagent detection. Also the minimal detectable amount for an acetylcholinesterase inhibitor tested was much less than that needed for the microplate assay. We screened 15 Amaryllidaceae extracts using this TLC method, and chose candidates for acetylcholinesterase inhibitor isolation.

Vitamin E improves microsomal phospholipase A2 activity and the arachidonic acid cascade in kidney of diabetic rats.

The purpose of the present study was to investigate the effects of vitamin E on microsomal phospholipase A2 activity and the arachidonic acid cascade in the kidneys of streptozotocin (STZ)-induced diabetic rats. Sprague-Dawley male rats weighing 100 +/- 10 g were randomly assigned to one normal and three STZ-induced diabetic groups. The diabetic groups were fed a vitamin E-free diet (the DM-0E group), 40 mg vitamin E/kg diet (the DM-40E group) or a 400 mg vitamin E/kg diet (the DM-400E group). The kidney vitamin E concentrations were 59 and 49% lower in the DM-0E and DM-40E groups, respectively, than in the normal group. The kidney thiobarbituric acid reactive substance concentrations in the DM-0E, DM-40E and DM-400E groups were 119, 84 and 33% greater, respectively, than that in the normal group. The concentration in the DM-400E group was 39% lower than that in the DM-0E group. The phospholipase A2 (PLA2) activity in the kidney microsomes of the DM-0E-40E and DM-400E groups were 88, 58 and 35% greater, respectively, than that in the normal group. The activity in the DM-400E group was 28% lower than that in the DM-0E group and 16% lower than that in the DM-40E group. The differences in the phospholipids in the kidney microsomes included reductions in the phosphatidylcholine and phosphatidylethanolamine compositions. Phosphatidylethanolamine hydrolysis in the kidney microsomes of the DM-0E and DM-40E groups were 84 and 64%, which did not differ from the DM-400E group. The formation of thromboxane A2 (TXA2) in the kidney microsomes was 137 and 70% greater in the DM-0E and DM-40E groups, respectively, than in the normal group. TXA2 formation did not differ between the DM-400E and normal groups. The formation of prostacyclin in the kidney microsomes was 60 and 44% lower in the DM-0E and DM-40E groups, respectively, than in the normal group, whereas the DM-400E group did not differ from that in the normal group. The ratio of prostacyclin to TXA2 was 82 and 65% lower than normal in the DM-0E and DM-40E groups, respectively. Kidney function appears to be improved by vitamin E supplementation due to its antithrombus action, which in turn controls the arachidonic acid cascade system.

Differential damage in bacterial cells by microwave radiation on the basis of cell wall structure.

Microwave radiation in Escherichia coli and Bacillus subtilis cell suspensions resulted in a dramatic reduction of the viable counts as well as increases in the amounts of DNA and protein released from the cells according to the increase of the final temperature of the cell suspensions. However, no significant reduction of cell density was observed in either cell suspension. It is believed that this is due to the fact that most of the bacterial cells inactivated by microwave radiation remained unlysed. Scanning electron microscopy of the microwave-heated cells revealed severe damage on the surface of most E. coli cells, yet there was no significant change observed in the B. subtilis cells. Microwave-injured E. coli cells were easily lysed in the presence of sodium dodecyl sulfate (SDS), yet B. subtilis cells were resistant to SDS.

CpG methylation is maintained in human cancer cells lacking DNMT1.

Hypermethylation is associated with the silencing of tumour susceptibility genes in several forms of cancer; however, the mechanisms responsible for this aberrant methylation are poorly understood. The prototypic DNA methyltransferase, DNMT1, has been widely assumed to be responsible for most of the methylation of the human genome, including the abnormal methylation found in cancers. To test this hypothesis, we disrupted the DNMT1 gene through homologous recombination in human colorectal carcinoma cells. Here we show that cells lacking DNMT1 exhibited markedly decreased cellular DNA methyltransferase activity, but there was only a 20% decrease in overall genomic methylation. Although juxtacentromeric satellites became significantly demethylated, most of the loci that we analysed, including the tumour suppressor gene p16INK4a, remained fully methylated and silenced. These results indicate that DNMT1 has an unsuspected degree of regional specificity in human cells and that methylating activities other than DNMT1 can maintain the methylation of most of the genome.

Chlorpromazine-induced inhibition of catecholamine secretion by a differential blockade of nicotinic receptors and L-type Ca2+ channels in rat pheochromocytoma cells.

We investigated the effect of chlorpromazine (CPZ), a phenothiazine neuroleptic, on catecholamine secretion in rat pheochromocytoma (PC12) cells. CPZ inhibited [3H]norepinephrine ([3H]NE) secretion induced by 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP), an agonist of nicotinic acetylcholine receptors (nAChRs) with an IC50 value of 1.0 +/- 0.2 microM. The DMPP-induced rise in cytosolic free Ca2+ concentration [Ca2+]i was inhibited by CPZ with an IC50 of 1.9 +/- 0.1 microM. The DMPP-induced increase in cytosolic free Na+ concentration [Na+]i was also inhibited by CPZ with a similar potency. Furthermore, the binding of [3H]nicotine to PC12 cells was inhibited by CPZ with an IC50 value of 2.7 +/- 0.6 microM, suggesting that the nAChRs themselves are inhibited by CPZ. In addition, both 70 mM K+-induced [3H]NE secretion and [Ca2+]i increase were inhibited by CPZ with IC50 of 7.9 +/- 1.1 and 6.2 +/- 0.3 microM, respectively. Experiments with Ca2+ channel antagonists suggest that L-type Ca2+ channels are mainly responsible for the inhibition. We conclude that CPZ inhibits catecholamine secretion by blocking nAChRs and L-type Ca2+ channels, with the former being more sensitive to CPZ.

Purification and characterization of brevinase, a heterogeneous two-chain fibrinolytic enzyme from the venom of Korean snake, Agkistrodon blomhoffii brevicaudus.

A fibrinolytic enzyme, designated as brevinase, was purified from the venom of Korean snake, Agkistrodon blomhoffii brevicaudus. Brevinase cleaved both the Aalpha- and Bbeta-chains of fibrinogen but did not affect the gamma-chain. It showed beta-fibrinogenase activity devoid of fibrinogen clotting and caseinolytic activity. The fibrinolytic activity was completely inhibited by PMSF, DFP, Pefabloc, and DTT, indicating brevinase is a serine protease requiring disulfide bridge(s) for its activity. It kept 80% of the initial activity after heating at 100 degrees C for 3 min, showed an equal maximum activity in the pH range from 5.5 to 8.5, and was inactivated by Zn(2+). Brevinase consists of two polypeptide chains of 16.5 and 17 kDa linked by disulfide bridge(s). The N-terminal amino acid sequences of 16.5 and 17 kDa chains showed homology to the N-terminal and the internal (central region) amino acid sequences of single-chain fibrinolytic enzymes in snake venom, respectively.

Changes of tissue factor activity on inflammatory stimulus and aging in rat.

Tissue factor (TF), a principal initiator of the vertebrate coagulation cascade, is expressed in organ tissues, cells and blood. TF is known to be induced in endothelial cells, monocytes and macrophages by inflammatory stimuli and in many pathologic conditions. By using the modified method for in vivo TF activity assay, we found that turpentine oil injection as an inflammatory stimulus also induced the TF activity in lung and brain tissues of rats. And the age-related increase in TF activity was observed in healthy rat brain tissue.

Mutational analysis of the fusion peptide of the human immunodeficiency virus type 1: identification of critical glycine residues.

The ability of human immunodeficiency virus type 1 (HIV-1) to fuse its membrane with the membrane of the target cell is a function of a approximately 23-amino-acid amino-terminal segment of the gp41 subunit of the envelope glycoprotein complex, known as the fusion peptide. The sequence of the fusion peptide is highly conserved among different variants of HIV-1 and is also very similar to that of HIV-2 and SIV. The fusion peptide is very hydrophobic and has a high content of glycine and alanine residues. Representation of the fusion peptide of HIV-1 as an alpha-helix predicts that most glycine residues would be found on one face of the alpha-helix. To assess the importance of the glycine residues for the fusogenic activity of the envelope glycoprotein complex, we mutagenized each glycine residue in the fusion peptide individually to a valine residue. The mutant envelope constructs were tested for their ability to induce syncytia (cell/cell fusion) and to mediate infection (virus/cell fusion) of CD4-positive cells. The results of our analyses show that two glycine residues (G10 and G13) located within the sequence FLGFLG in the middle of the fusion peptide are critical for syncytium formation and for the establishment of a productive infection, whereas other glycine residues (G3, G5, and G20) are more permissive to substitutions. Mutation of each of the two phenylalanines (F8 and F11) of the FLGFLG sequence to valine also decreased fusion, although to a lesser extent than mutation of G10 and G13. These observations demonstrate that G10 and G13 are critical elements of the fusion peptide and suggest that, in addition to hydrophobicity, the exact amino acid composition and structure of the fusion peptide are critical for function.

Interaction of tyrosine-based sorting signals with clathrin-associated proteins.

Tyrosine-based signals within the cytoplasmic domain of integral membrane proteins mediate clathrin-dependent protein sorting in the endocytic and secretory pathways. A yeast two-hybrid system was used to identify proteins that bind to tyrosine-based signals. The medium chains (mu 1 and mu 2) of two clathrin-associated protein complexes (AP-1 and AP-2, respectively) specifically interacted with tyrosine-based signals of several integral membrane proteins. The interaction was confirmed by in vitro binding assays. Thus, it is likely that the medium chains serve as signal-binding components of the clathrin-dependent sorting machinery.

The mid-trimester placenta previa: a prospective follow-up.

One hundred and fifty-six mid-trimester sonograms were performed at our prenatal diagnostic unit. Twenty women were found to have a low-lying placenta or placenta previa and were followed by serial ultrasound examinations to observe changes in placental position. Eighty percent of women, i.e., 16/20, with a low-lying placenta had converted to normal implantation by the time of delivery. Most of the conversions had taken place at approx. 34 weeks of gestation. The patients with mid-trimester low-lying placenta had an increased risk of third-trimester bleeding, abruptio placentae and cesarean sections. The infants were also at risk of premature delivery. Patients with mid-trimester low-lying or placenta previa should be followed by ultrasound to monitor delivery.

Metallothioneins with interdomain hinges expanded by insertion mutagenesis.

Specific peptides of varying lengths were inserted between the two metal cluster domains of metallothionein (MT), which normally are spanned by only three amino acids, Lys-Lys-Ser. These interdomain expansions were made to test if such structural alterations would affect MT function. These constructs were engineered by inserting defined oligonucleotides of up to four tandem repeats of dodecanucleotides and hexanucleotides into an Alu-1 endonuclease cleavage site, which separates the two exonic regions of an MT-coding sequence from Chinese hamster ovary cells, MT-2. The native and altered sequences were cloned into a high expression Escherichia coli-yeast shuttle vector and used to transform yeast cells whose endogenous MT genes had been previously deleted. Using metal resistance as a biological marker, all constructs were shown to be functional in rendering the host cells resistant to either copper or cadmium. As the inserts, by nature of their amino acid sequence, could add flexibility to the otherwise compact molecule, the two domains apparently are active independently. The level of activity, however, diminished with the length of the insert. Determinations for copy number of the chimeric plasmids and MT mRNAs in the transformed cells showed that the replicational and transcriptional capacity of the long and short constructs were equivalent.