Collagen glucosyl- and galactosyltransferases of cultured human fetal lung fibroblasts

Collagen-galactosyltransferase and collagen-glucosyltransferase activities have been studied in cultured human fetal lung WI-38 and IMR-90 diploid fibroblasts. These enzymes functioned in concert to synthesize glucosylgalactosylhydroxylysine units as found naturally in collagens, basement membranes, and certain serum glycoproteins. The transferases used UDP-Gal and UDP-Glc as glycose donors, collagens and collagen-derived peptides or glycopeptides as glycose acceptors, and worked best in the presence of manganese as a required divalent cation. Two pH optima, between pH 6 and 6.5 and between pH 7.5 and 8, were noted for each type of transferase, and these optima, particularly in the case of glucosyltransferase, were evident regardless of size of acceptor employed in the assay. About 35% of the total activity of each enzyme was found in the soluble fractions of cell homogenates, and, of the particulate fraction activities, about 50% could be released by mild sonication or by treatment with Triton X-100. Assessment of transferase activities as a function of cellular aging in culture revealed that significant decreases in enzyme levels occurred as the cell approached senescence (late Phase II), and these effects were reversed when cells attained senescence (Phase III). Addition of ascorbic acid to young cultures, under conditions known to increase endogenous collagenpeptide hydroxylation, caused no effects on the activities of the glycosyltransferases toward exogenous acceptors. These results suggested that the activities of collagen-hydroxylases and glycosyltransferase might not be coordinately regulated, and that, regardless of the hydroxylation events, glycosylation of the peptide might be limited to a specific fraction of hydroxylysine residues during the post-translational modification of collagen.     

Novel surface lipids of diapausing Manduca sexta pupae. Long chain oxoalcohol esters of acetoacetic, hydroxybutyric, and acetic acids

Ester components in the surface wax from diapausing tobacco hornworm pupae, Manduca sexta L., were separated by thin layer chromatography and gas-liquid chromatography, and characterized by infrared spectroscopy and gas-liquid chromatography-mass spectrometry. Three groups of esters were identified as natural derivatives of acetic acid, acetoacetic acid, and 3-hydroxybutyric acid. The major ester fraction was identified as a mixture of C26 (10%), C27 (5%), and C28 (85%) oxoalcohol esters of acetoacetic acid. The major homolog consisted of equal amounts of 11-oxooctacosanyl 3-oxobutanoate and 12-oxooctacosanyl 3-oxobutanoate. Lesser amounts of 11- and 12-oxooctacosanyl and n-octacosanyl esters of acetic and 3-hydroxybutyric acids were also identified. The chain length distributions of these C26, C27, and C28 oxoalcohol and n-primary alcohol ester moieties, as well as the isomeric ratios for the 11- and 12-oxoalcohol isomers, were similar to the oxoaldehydes and unesterified oxoalcohols previously identified by Buckner et al (Buckner, J. S., Nelson, D. R., Haak, H., and Pomonis, J. G. (1984) J. Biol. Chem. 259, 8452-8470) as lipid components of the surface wax of M. sexta pupae.     

Accumulation of malto-oligosaccharides in the syncytiotrophoblastic cells of first-trimester human placentas.

A cell-surface microvillar fraction that was isolated from the syncytiotrophoblastic cells of first-trimester human placentas was found to contain very high concentrations (890 +/- 32 microgram of hexose/mg of protein) of a class of low-molecular-weight oligosaccharides that were comprised entirely of glucose. T.l.c. and gel filtration showed that the saccharides contained from one to six glucose residues. The structures of the most prominent members of the series, a tetra- and a tri-saccharide, were determined. The anomeric configuration of the glucose residues was alpha, and methylation linkage analysis gave terminal and 4-linked hexose residues. These malto-oligosaccharides contained one reducing terminus per molecule, indicating that they were free and not bound to other structural elements of the cells. Within the placenta they appeared to be concentrated in the first-trimester trophoblastic cells, since crude membrane and particulate fractions isolated from either term trophoblastic cells or cultured placental fibroblasts did not contain detectable amounts of glucose oligomers. This series of oligosaccharides was similar to the products that are formed when glycogen is degraded by alpha-amylase in liver homogenates and may be indicative of a similar, highly active enzymic reaction closely associated with the brush border of the syncytiotrophoblastic cells of the first-trimester human placenta. Although the role of these oligosaccharides remains obscure they are probably involved in foetal metabolism.     

Screening of Drug Metabolizing Enzymes for the Ginsenoside Compound K In Vitro: An Efficient Anti-Cancer Substance Originating from Panax Ginseng

Ginsenoside compound K (CK), a rare ginsenoside originating from Panax Ginseng, has been found to possess unique pharmacological activities specifically as anti-cancers. However, the role of cytochrome P450s (CYPs) in the metabolism of CK is unclear. In this study, we screened the CYPs for the metabolism of CK in vitro using human liver microsomes (HLMs) or human recombinant CYPs. The results showed that CK inhibited the enzyme activities of CYP2C9 and CYP3A4 in the HLMs. The K_m and V_max values of CK were 84.20±21.92 μM and 0.28±0.04 nmol/mg protein/min, respectively, for the HLMs; 34.63±10.48 μM and 0.45±0.05 nmol/nmol P450/min, respectively, for CYP2C9; and 27.03±5.04 μM and 0.68±0.04 nmol/nmol P450/min, respectively, for CYP3A4. The IC₅₀ values were 16.00 μM and 9.83 μM, and K_i values were 14.92 μM and 11.42μM for CYP2C9 and CYP3A4, respectively. Other human CYP isoforms, including CYP1A2, CYP2A6, CYP2D6, CYP2E1, and CYP2C19, showed minimal or no effect on CK metabolism. The results suggested that CK was a substrate and also inhibitors for both CYP2C9 and CYP3A4. Patients using CK in combination with therapeutic drugs that are substrates of CYP2C9 and CYP3A4 for different reasons should be careful, although the inhibiting potency of CK is much poorer than that of enzyme-specific inhibitors.     

Noni as an anxiolytic and sedative: A mechanism involving its gamma-aminobutyric acidergic effects

Noni (Morinda citrifolia) is increasing in worldwide popularity as a food or dietary supplement with versatile health benefits. The aim of this study was to investigate the effects of Noni fruit on anxiety symptoms in vitro. To this end, a competitive GABAa receptor-binding assay was developed. Our preliminary study indicates that the methanol crude extract of Noni fruit showed significant affinity to the gamma-aminobutyric acid A (GABAa) inhibitory neurotransmitter receptors, and displayed 75% binding inhibition of the agonist radioligand [3H] muscimol at a concentration of 100 microg/ml. Further experiments demonstrated that the MeOH extract, and its BuOH and H2O partitions, exhibited IC50 values of 22.8, 27.2, and 17.1 microg/ml, respectively, in the GABAa-binding assay. Experimental results with Noni fruit indicate the presence of competitive ligand(s), which may bind to the GABAa receptor as an agonist, and thus induce its anxiolytic and sedative effects. The study provides an in vitro rationale for one of Noni's versatile and traditional uses. In addition, an HPLC fingerprint profile of the methanolic extract of Noni fruit has been established for quality control purpose.     

The distribution of marked dermal cells from small localized implants in limb regenerates

Numerous experiments have demonstrated that skin has a profound influence on the pattern of limb regeneration in urodeles. In this investigation, the fate during regeneration of marked cells derived from narrow strips of skin inserted into different positions around the limb circumference has been followed. Skin strips were taken from triploid axolotls and transplanted into diploid sibling animals. The distribution of trinucleolate cells was determined at the site of amputation and in the regenerated limb. The results indicate that at the time of amputation marked cells appear to be localized to the graft, whereas in the regenerated marked cells may be found at all proximal-distal levels and at any position around the circumference of the limb. These results are discussed in terms of a possible mechanism for distal outgrowth.     

In vitro stabilization of 6-methylsalicylic acid synthetase from Penicillium urticae

In continuing studies of patulin biosynthesis, the first enzyme of the pathway, 6-methylsalicylic acid synthetase, was found to be far more labile than were the later enzymes of the pathway. Attempts were made to stabilize 6-methylsalicylic acid synthetase in vitro. The combined addition of the cofactor NADPH, the substrates acetyl-CoA and malonyl-CoA, the reducing agent dithiothreitol, and the proteinase inhibitor phenylmethylsulfonyl fluoride to cell-free extracts was found to prolong the half-life of the enzyme as much as 12-fold. This suggested that proteolysis and the conformational integrity of the enzyme may play an important role in controlling the duration of antibiotic biosynthesis in vivo. This was in agreement with the finding that the intracellular proteinase content of antibiotic-producing cells of Penicillium urticae rapidly increased just before the loss of 6-methylsalicylic acid synthetase content. These in vitro stabilization studies have provided some insight into the metabolic conditions that may stabilize these enzymes in vivo, and into possible ways of extending the life of these catalysts.