Structure of human alpha1-acid glycoprotein and its high-affinity binding site

Secondary and tertiary structures of human blood alpha(1)-acid glycoprotein, a member of the lipocalin family, have been studied for the first time by infrared and Raman spectroscopies. Vibrational spectroscopy confirmed details of the secondary structure and the structure content predicted by homology modeling of the protein moiety, i.e., 15% alpha-helices, 41% beta-sheets, 12% beta-turns, 8% bands, and 24% unordered structure at pH 7.4. Our model shows that the protein folds as a highly symmetrical all-beta protein dominated by a single eight-stranded antiparallel beta-sheet. Thermal dynamics in the range 20-70 degrees C followed by Raman spectroscopy and analyzed by principle component analysis revealed full reversibility of the protein motion upon heating dominated by decreasing of beta-sheets. Raman difference spectroscopy confirmed the proximity of Trp(122) to progesterone binding.     

The inhibitory effects of flavonoids and antiestrogens on the Glut1 glucose transporter in human erythrocytes

Flavonoids and isoflavonoids are potent inhibitors of glucose efflux in human erythrocytes. Net changes of sugars inside the cells were measured by right angle light scattering. The inhibitory potency of hydroxylated flavonoids depends on the pH of the medium. The apparent affinity is maximal at low pH where the molecule is in the undissociated form. The following K(i)-values at pH 6.5 in microM have been obtained: phloretin 0.37+/-0.03, myricetin 0.76+/-0.42, quercetin 0.93+/-0.28, kaempferol 1.33+/-0.17, isoliquiritigenin 1.96, genistein 3.92+/-0.62, naringenin 8.88+/-1.88, 7-hydroxyflavone 17.58+/-3.15 and daidzein 18.62+/-2.85. Flavonoids carrying hydroxyl groups are weak acids and are deprotonated at high pH-values. From spectral changes pK-values between 6.80 (naringenin) and 7.73 (myricetin) have been calculated. No such pK-value could be obtained from quercetin which was rather unstable at alkaline pH. Flavone itself without a hydroxyl group does not demonstrate any absorbance changes at different pH-values and no significant change in inhibition of glucose transport with pH (K(i)-value around 35 microM). In this respect it is similar to the antiestrogens diethylstilbestrol, tamoxifen and cyclofenil with K(i)-values for glucose efflux inhibition of 2.61+/-0.30, 6.75+/-2.03 and 3.97+/-0.54 microM. Except for phloretin, the flavonoids investigated have planar structures. The inhibitory activity in glucose efflux of planar flavonoids increases exponentially with the number of hydroxyl groups in the molecule.     

Shmuel Shaltiel

The ability of cells to synthesize and secrete proteins is essential for numerous cellular functions. Therefore, when mutations in one component of the secretory pathway result in a tissue-specific defect, a unique opportunity arises to examine the molecular mechanisms at play. The recent finding that a defect in the protein sedlin, whose yeast counterpart is involved in the first step of the secretory pathway, leads to a cartilage-specific disorder in humans raises numerous questions and interesting possibilities for understanding both the pathobiology involved and the role of membrane traffic in normal cartilage development.     

Pre-treatment of Caco-2 cells with zinc during the differentiation phase alters the kinetics of zinc uptake and transport(2)

The Caco-2 cell model was used to study the efficiency of absorption and endogenous excretion of zinc (Zn) regulated by dietary Zn concentration. Cells were seeded onto high pore-density membranes and maintained in medium supplemented with 10% FBS. After confluence, cells were treated with 5 or 25 μmol Zn/L for 7 d, and Zn uptake and transport were measured in both apical (AP) and basolateral (BL) directions by using ⁶⁵Zn. Similar cells were labeled with ⁶⁵Zn and the release of Zn to the AP and BL sides was measured. The AP uptake of Zn in cells exposed to 25 μmol Zn/L was slower (p < 0.05) than that in cells exposed to 5 μmol Zn/L. The AP to BL transport rate in the 25 μmol Zn/L group was only 40% (p < 0.05) of that in the 5 μM group. In contrast, the rate of BL Zn uptake was 4-fold higher in cells treated with 25 μmol Zn/L than in those treated with 5 μmol Zn/L (p < 0.05). The BL to AP transport rate was 2-fold higher in cells treated with 25 μmol Zn/L than in those treated with 5 μmol Zn/L (p < 0.05). Basolateral uptake was 6 to 25 times greater (p < 0.05) than AP uptake for cells treated with 5 and 25 μmol Zn/L, respectively. The rate of Zn release was enhanced about 4-fold (p < 0.05) by 25 μmol Zn/L treatment. Release to the BL side was 10 times greater than to the AP side. Zn-induced metallothionein (MT), thought to down-regulate AP to BL Zn transport, was 4-fold higher (p < 0.001) in the 25 μmol Zn/L group than in the 5 μM group, but the rate of BL Zn release was higher in cells treated with 25 μmol Zn/L than in those treated with 5 μmol Zn/L (p < 0.05). Induced changes in transport rates by media Zn concentrations could involve the up- and/or down-regulation of Zn influx and efflux proteins such as the ZIP and ZnT families of Zn transporters.     

Importance of direct determination of amino acid co- and counter-transport stoichiometries

Summary. The stoichiometry of amino acid transport with co- or counter-substrates of a given system has been found to vary with the amino acid species. This phenomenon has been studied directly in only a few cases, however, by measuring the fluxes of the substrates simultaneously. More frequently, the apparent transport stoichiometries of single amino acid species with co- or counter-substrates are estimated indirectly by thermodynamic criteria or cooperative kinetic effects. Unfortunately the latter indirect mea-sures of apparent stoichiometry often yield different results than direct measurement of simultaneous fluxes. These differences often cannot be explained by uncoupled transport of one of the co- or counter-substrates or by other characteristics of the transport process that would make the direct measurement of stoichiometry inaccurate. For these reasons, investigators are encouraged to measure the stoichiometry of transport directly by measuring simultaneous fluxes of co- and counter-substrates. Indirect measures of apparent stoichiometry may, however, reflect important details of a transport mechanism even if they are inconsistent with the actual stoichiometry of transport. Received February 20, 2000 Accepted May 18, 2000     

The control of the proventriculus in the honeybee (Apis mellifera carnica L.) II. Feedback mechanisms

The mechanisms underlying the control of solution transport rates through the proventriculus in foraging honeybees were investigated in individuals trained to collect defined amounts of sugar solutions. Following feeding, bees were injected either with metabolisable (glucose, fructose, trehalose), or non-metabolisable (sorbose) sugars, in order to distinguish between haemolymph osmolarity and haemolymph sugar levels as factors controlling the solution transport rates through the proventriculus. After a fixed period, workers were dissected in order to measure crop content and haemolymph sugar titers. Between feeding and dissection, the metabolic rate of every investigated forager was measured using open-flow respirometry. Bees injected with metabolisable sugars 15 min after feeding were observed to reduce their solution transport rates through the proventriculus, but injection of non-metabolisable sugars had no influence on them. This suggests that the solution transport rate through the proventriculus is controlled by the concentration of metabolisable compounds in the haemolymph, and not by the haemolymph osmolarity. A period of 10 min after injection of metabolisable sugars was enough to observe reduced solution transport rates. However, if bees were injected only 5 min after feeding, no reduced solution transport rates were observed 10 min after injection.     

Vitamin K uptake in hepatocytes and hepatoma cells

Hepatocellular carcinoma (HCC) or hepatoma cells have impaired ability to perform vitamin K-dependent carboxylation reactions. Vitamin K can also inhibit growth of HCC cells in vitro. Both carboxylation and growth inhibition are vitamin K dose dependent. We used rat hepatocytes, a vitamin K-growth sensitive (MH7777) and a vitamin K-growth resistant (H4IIE) rat hepatoma cell line to examine vitamin K uptake and vitamin K-mediated microsomal carboxylation. We found that vitamin K is taken up by normal rat hepatocytes against a saturable concentration gradient. The relative rates of uptake by rat hepatocytes and the two rat cell lines MH7777 and H4IIE correlated with their sensitivity to vitamin K-mediated cell growth inhibition. Pooled hepatocytes from liver nodules from rats treated with the hepatocarcinogen diethylnitrosamine (DEN) also had a reduced rate of vitamin K uptake. However, using a cell-free system, microsomes from both normal rat hepatocytes and the two rat hepatoma cell lines had a similar ability to support carboxylation mediated by exogenously added vitamin K. The results support the hypothesis that different sensitivity of hepatoma cells to vitamin K may be due to differences in vitamin K uptake and may be unrelated to the actions of vitamin K on carboxylation.     

Human brain nucleoside diphosphate kinase activity is decreased in Alzheimer's disease and Down syndrome

In brain, nucleoside diphosphate kinase (NDPK) and its coding gene, nm23, have been implicated to modulate neuronal cell proliferation, differentiation, and neurite outgrowth. However, a role of NDPK in neurodegenerative diseases has not been reported yet. Using proteomics techniques, we evaluated the protein levels of NDPK-A in seven brain regions from patients with Alzheimer's disease (AD) and Down syndrome (DS) showing AD-like neuropathology. NDPK-A was significantly decreased in brain regions (frontal, occipital, and parietal cortices) of both disorders. Due to the limitation of brain samples, the activity of NDPK was measured in three brain regions (frontal cortex, temporal cortex, and cerebellum). The specific activity of NDPK was significantly decreased in AD (frontal cortex) and DS (frontal and temporal cortices). Since NDPK-B could also drive the activity of NDPK, protein expression levels of both NDPK-A and NDPK-B were studied in frontal cortex by Western blot analysis. NDPK-A was significantly decreased in AD, which was consistent with the results of proteomics. However, NDPK-A was slightly decreased in DS and protein expression levels of NDPK-B in both DS and AD were moderately decreased, without reaching statistical significance. We propose that oxidative modification of NDPK could lead to the decreased activity of NDPK and, subsequently, influence several neuronal functions in neurodegenerative diseases as multifunctional enzyme through several mechanisms.     

Transport of cadmium across the apical membrane of epithelial cell lines

Cadmium (Cd) uptake and secretion across the apical membrane of epithelial cells was studied using LLC-PK1 cells cultured on Petri dishes and permeable membranes, respectively. Cd accumulation in cells from the apical medium was decreased by low temperature and metabolic inhibitors. A saturable tendency was observed between initial Cd accumulation and increased concentrations of Cd in the apical medium at 37 degrees C, but not at 4 degrees C. Co-incubation with ZnCl2 or CuCl2 competitively decreased Cd accumulation at 37 degrees C. A decrease in the pH of the apical medium markedly decreased Cd accumulation. Pretreatment of cells with an inorganic anion-exchange inhibitor significantly decreased Cd uptake at pH 7.4 in the presence of bicarbonate, but only marginally in its absence. A decrease in the pH of the apical medium increased the secretory (basolateral-to-apical) transport of Cd, with a concomitant decrease in the cellular accumulation of Cd. Co-incubation with Cd and tetraethylammonium, a typical substrate of the organic cation transporter, decreased Cd transport, with a concomitant increase in cellular Cd accumulation. The uptake and secretion of Cd across the apical membrane appear to be partly mediated via an inorganic anion exchanger and a H+ antiport of the organic cation transport system, respectively. Therefore, a decrease in pH of the apical medium markedly decreases Cd accumulation, possibly as a result of not only the decrease in Cd uptake via an inorganic anion exchanger, but also the increase in Cd secretion via the Cd2+/H+ antiport. Further evidence of the antiport was obtained from experiments using brush border membrane vesicles isolated from rat kidney and small intestine. In addition, passive diffusion of Cd appears to be decreased by low temperature and a decrease in pH.