Elevated serum α-fetoprotein level of nude mice bearing hepatoma cells by treatment with monoclonal antibodies to α-fetoprotein

This study was carried out to clarify the reason for elevation of serum α-fetoprotein (AFP) level of nude mice bearing hepatoma cells after treatment with monoclonal antibodies (MoAbs) to AFP. MoAbs to AFP showed no effect on the cumulative amounts of AFP secreted from human hepatoma cell line, HuH-7, in vitro. However, the treatment of nude mice bearing HuH-7N cells (HuH-7 xenograft) with MoAbs to AFP led to elevation of the serum AFP level in spite of the fact that the growth curve of HuH-7N cells was similar to that for PBS treatment. This apparent elevation of the serum AFP level is thought to be due to the slow elimination of AFP-MoAb immune complexes with little lattice structure from circulation, but not the enhancement of AFP secretion of HuH-7N cells. Thus, when using a MoAb alone or MoAb-drug conjugate, the serum AFP level should only be cautiously used as a tumor marker for evaluating the targeting immunotherapy.     

Five New Phenolic Glycosides from Viburnum luzonicum

Viburnum luzonicum is widely distributed in China. Its branch extracts showed potential α-amylase and α-glucosidase inhibitory activities. In order to discover new bioactive constituents, five undescribed phenolic glycosides, viburozosides A−E ( 1 – 5 ), were obtained by bioassay-guided isolation coupled with HPLC-QTOF-MS/MS analysis. Their structures were elucidated by spectroscopic analyses, including 1D NMR, 2D NMR, ECD, and ORD. All compounds were tested for their α-amylase and α-glucosidase inhibitory potency. Compound 1 showed significantly competitive inhibition against α-amylase (IC₅₀=17.5 μM) and α-glucosidase (IC₅₀=13.6 μM).     

Phytoconstituents of Selaginella effusa Alston and Their α-Glucosidase as well as Urease Inhibitory Activities

Three new compounds ( 1 – 2 , 14 ), as well as 22 known compounds ( 3 – 13 , 15 – 25 ), were extracted for the first time from the Selaginella effusa Alston (S. effusa). For the unknown compounds, the planar configurations were determined via NMR and by high-resolution mass spectrometry, while their absolute configurations were determined by calculated electronic circular dichroism (ECD), and the configuration of the stereogenic center of biflavones 4 – 5 were established for the first time. The pure compounds ( 1 – 25 ) were tested in vitro to determine the inhibitory activity of the enzyme-catalyzed reactions. Compounds 1 – 9 inhibited α-glucosidase with IC₅₀ values ranging from 0.30±0.02 to 4.65±0.04 μM and kinetic analysis of enzyme inhibition indicated that biflavones 1 – 3 were mixed-type α-glucosidase inhibitors. Compounds 12 – 13 showed excellent inhibitory activity against urease, with compound 12 (IC₅₀=4.38±0.31 μM) showing better inhibitory activity than the positive control drug AHA (IC₅₀13.52±0.61 μM). In addition, molecular docking techniques were used to simulate inhibitor-enzyme binding and to estimate the binding posture of the α-glucosidase and urease catalytic sites.     

CX3CL1 induces cell migration and invasion through ICAM-1 expression in oral squamous cell carcinoma cells

Human oral squamous cell carcinoma (OSCC) has been associated with a relatively low survival rate over the years and is characterized by a poor prognosis. C-X3-C motif chemokine ligand 1 (CX3CL1) has been involved in advanced migratory cells. Overexpressed CX3CL1 promotes several cellular responses related to cancer metastasis, including cell movement, migration and invasion in tumour cells. However, CX3CL1 controls the migration ability, and its molecular mechanism in OSCC remains unknown. The present study confirmed that CX3CL1 increased cell movement, migration and invasion. The CX3CL1-induced cell motility is upregulated through intercellular adhesion molecule-1 (ICAM-1) expression in OSCC cells. These effects were significantly suppressed when OSCC cells were pre-treated with CX3CR1 monoclonal antibody (mAb) and small-interfering RNA (siRNA). The CX3CL1-CX3CR1 axis activates promoted PLCβ/PKCα/c-Src phosphorylation. Furthermore, CX3CL1 enhanced activator protein-1 (AP-1) activity. The CX3CR1 mAb and PLCβ, PKCα, c-Src inhibitors reduced CX3CL1-induced c-Jun phosphorylation, c-Jun translocation into the nucleus and c-Jun binding to the ICAM-1 promoter. The present results reveal that CX3CL1 induces the migration of OSCC cells by promoting ICAM-1 expression through the CX3CR1 and the PLCβ/PKCα/c-Src signal pathway, suggesting that CX3CL1-CX3CR1-mediated signalling is correlated with tumour motility and appealed to be a precursor for prognosis in human OSCC.     

Role of the outer tissue in abscisic acid-mediated growth suppression of etiolated squash hypocotyl segments

Exogenously applied abscisic acid (ABA) substantially suppressed the elongation of hypocotyl segments of etiolated squash ( Cucurbita maxima Duch. cv. Houkou-Aokawaamaguri) after a 3 h lag period, without changes in the osmolalities of the apoplastic and symplastic solutions in the segment.
Segments with the outer tissues removed elongated more rapidly than unpeeled segments (whole segments). ABA did not suppress the elongation of peeled segments. When the segments were incubated in [¹⁴C]-glucose, radioactivity was more effectively incorporated into the cell wall fractions of the outer than into those of the inner tissue. ABA significantly inhibited the incorporation of radioactivity into hermicellulose and cellulose of the outer tissue prior to the suppression of segment elongation, but it did not inhibit the incorporation into the pectic traction of the outer tissue or into any of the cell wall fractions of the inner tissue. These results indicate that ABA primarily affected the outer tissue, in which it specifically reduced the synthesis of hemicellulose and cellulose prior to the ABA-mediated suppression of growth.     

Effects of detergents on the secondary structures of prion protein peptides as studied by CD spectroscopy.

Pathogenic prion proteins (PrP(Sc)) are thought to be produced by alpha-helical to beta-sheet conformational changes in the normal cellular prion proteins (PrP(C)) located solely in the caveolar compartments. In order to inquire into the possible conformational changes due to the influences of hydrophobic environments within caveolae, the secondary structures of prion protein peptides were studied in various kinds of detergents by CD spectra. The peptides studied were PrP(129-154) and PrP(192-213); the former is supposed to assume beta-sheets and the latter alpha-helices, in PrP(Sc). The secondary structure analyses for the CD spectra revealed that in buffer solutions, both PrP(129-154) and PrP(192-213) mainly adopted random-coils (approximately 60%), followed by beta-sheets (30%-40%). PrP(129-154) showed no changes in the secondary structures even in various kinds of detergents such as octyl-beta-D-glucopyranoside (OG), octy-beta-D-maltopyranoside (OM). sodium dodecyl sulfate (SDS), Zwittergent 3-14 (ZW) and dodecylphosphocholine (DPC). In contrast, PrP(192-213) changed its secondary structure depending on the concentration of the detergents. SDS, ZW, OG and OM increased the alpha-helical content, and decreased the beta-sheet and random-coil contents. DPC also increased the alpha-helical content, but to a lesser extent than did SDS, ZW, OG or OM. These results indicate that PrP(129-154) has a propensity to adopt predominantly beta-sheets. On the other hand, PrP(192-213) has a rather fickle propensity and varies its secondary structure depending on the environmental conditions. It is considered that the hydrophobic environments provided by these detergents may mimic those provided by gangliosides in caveolae, the head groups of which consist of oligosaccharide chains containing sialic acids. It is concluded that PrP(C) could be converted into a nascent PrP(Sc) having a transient PrP(Sc) like structureunder the hydrophobic environments produced by gangliosides.     

Identification and partial characterization of α2-macroglobulin from the tuatara (Sphenodon punctatus)

α₂-Macroglobulin (α₂-M), a large molecular mass proteinase-binding protein, was identified in plasma from tuatara (Sphenodon), a rare reptile endemic to New Zealand. In this genus, α₂-M constitutes 11–13% of total plasma protein (∼2.2–3.9 mg/ml). Analysis of blood samples collected at approximately monthly intervals from individual tuatara indicated that the plasma level of α₂-M remains fairly constant. The subunits of tuatara α₂-M have an apparent molecular mass of ∼160 kDa as determined by SDS-polyacrylamide gel electrophoresis and the intact protein is an oligomer that contains inter-chain disulfide bonds. N-terminal sequence analyses of tuatara α₂-M revealed a distinct similarity to α-macroglobulins of other vertebrates and that at least two types of α₂-M subunits are present in plasma of tuatara.     

PLD activation in Chinese hamster ovary (CHO) cells transfected with PGF2α receptor cDNA

Pertussis toxin-insensitive GTP-binding protein was observed to be involved in prostaglandin F2α(PGF2α)-induced phosphoinositide metabolism in Chinese hamster ovary (CHO) cells transfected with PGF2α receptor cDNA (CHO-PGF2α·R cells) (Ito, S. et al. Biochem. Biophys. Res. Commun. 200: 756, 1994). In the present study, we investigated PGF2α-induced PLD activation in CHO-PGF2α·R cells. PLD activation was examined by measuring the production of [³H]phosphatidylbutanol ([³H]PBut), a specific product of the PLD-catalyzed transphosphatidylation reaction. PGF2α-induced [³H]PBut formation was concentration-dependent with the maximal level obtained at 1 μM PGF2α. The maximal [³H]PBut formation was observed at 2 min after addition of PGF2α. Depletion of extracellular Ca²⁺ with EGTA suppressed PGF2α-induced PLD activation by 50%. PKC inhibitors Ro31–8425 and calphostin C inhibited PGF2α-induced [³H]PBut formation by 50%. PTK inhibitors genistein and herbimycin A failed to inhibit PGF2α-induced PLD activation. A combination of maximal effective concentrations of PGF2α (1 μM) and PMA (100 nM) enhanced PLD activation in an additive manner. Pretreatment of the cells with PMA for 2 h down-regulated PKCα and decreased PGF2α-induced PLD activation. These results suggest that PLD activation by PGF2α is mediated by both PKC-dependent and -independent pathways and that PKCα is involved in the former pathway.     

Glutamine metabolism in AS-30D hepatoma cells. Evidence for its conversion into lipids via reductive carboxylation

A study was undertaken to assess the role of a physiological concentration of glutamine in AS-30D cell metabolism. Flux of¹⁴C-glutamine to¹⁴CO₂ and of¹⁴C-acetate to glutamate was detected indicating reversible flux between glutamate and TCA cycle -ketoglutarate. These fluxes were transaminase dependent. A flux analysis was compared using data from three tracers that label -ketoglutarate carbon 5, [2-¹⁴C]glucose, [1-¹⁴C]acetate and [5-¹⁴C]glutamine. The analysis indicated that the probability of flux of TCA cycle -ketoglutarate to glutamate was, at minimum, only slightly less than the probability of flux of -ketoglutarate through -ketoglutarate dehydrogenase. The apparent Km for oxidative flux of [¹⁴C]glutamine to¹⁴CO₂, 0.07 mM, indicated that this flux was at a maximal rate at physiological, 0.75 mM, glutamine. Although oxidative flux through -ketoglutarate dehydrogenase was the major fate of glutamine, flux of glutamine to lipid via reductive carboxylation of -ketoglutarate was demonstrated by measuring incorporation of [5-¹⁴C]glutamine into¹⁴C-lipid. In media containing glucose (6 mM), and glutamine (0.75 mM) 47 per cent of the lipid synthesized from substrates in the media was derived from glutamine via reductive carboxylation and 49 per cent from glucose. These findings of nearly equal fluxes suggest that lipogenesis via reductive carboxylation may be an important role of glutamine in hepatoma cells.