Comparison of Recombinant Human Haptocorrin Expressed in Human Embryonic Kidney Cells and Native Haptocorrin

Haptocorrin (HC) is a circulating corrinoid binding protein with unclear function. In contrast to transcobalamin, the other transport protein in blood, HC is heavily glycosylated and binds a variety of cobalamin (Cbl) analogues. HC is present not only in blood but also in various secretions like milk, tears and saliva. No recombinant form of HC has been described so far. We report the expression of recombinant human HC (rhHC) in human embryonic kidney cells. We purified the protein with a yield of 6 mg (90 nmol) per litre of cell culture supernatant. The isolated rhHC behaved as native HC concerning its spectral properties and ability to recognize both Cbl and its baseless analogue cobinamide. Similar to native HC isolated from blood, rhHC bound to the asialoglycoprotein receptor only after removal of terminal sialic acid residues by treatment with neuraminidase. Interestingly, rhHC, that compared to native HC contains four excessive amino acids (…LVPR) at the C-terminus, showed subtle changes in the binding kinetics of Cbl, cobinamide and the fluorescent Cbl conjugate CBC. The recombinant protein has properties very similar to native HC and although showing slightly different ligand binding kinetics, rhHC is valuable for further biochemical and structural studies

Two-step activation prodrugs: transplatin mediated binding of chemotherapeutic agents to vitamin B-12

Clinically approved organic chemotherapeutic drugs such as cytarabine, dacarbazine and anastrozole were attached to B12via a {CN-trans-Pt(NH3)2}-bridge to yield [{Co}-CN-{trans-Pt(NH3)2}-{drug}](2+). The active organic drugs are protected by the platinum complex and by B12, which represents at the same time the targeting vector. We refer to these bioconjugates as two-step activation prodrugs since two reactions are finally required to liberate the actual organic drugs. All three prodrugs are soluble and stable in water. The physiological stability and the therapeutic efficiency of [{Co}-CN-{trans-Pt(NH3)2}-{cytarabine}](2+) (2) were studied. Under physiological conditions, 2 is stable for 3 days. Its affinity to the cobalamin transport proteins (haptocorrin, intrinsic factor and transcobalamin) is not substantially affected despite the introduction of a bulky group in the β-axial position. The cleavage of the [trans-CN-Pt(NH3)2-{cytarabine}](+) complex was observed upon chemical reduction of Co(III)→ Co(II) with Zn(0). Cytarabine was subsequently released from the cleaved complex to exhibit its cytotoxicity. 2 displayed a reduced cytotoxicity (IC50 = 230 ± 62 nM) as compared to cytarabine (IC50 = 30 ± 5 nM). However, cytarabine released from 2 showed comparable cytotoxicity (IC50 = 30 ± 11 nM)

Cannabinoid receptor trafficking in peripheral cells is dynamically regulated by a binary biochemical switch

The cannabinoid G protein-coupled receptors (GPCRs) CB₁ and CB₂ are expressed in different peripheral cells. Localization of GPCRs in the cell membrane determines signaling via G protein pathways. Here we show that unlike in transfected cells, CB receptors in cell lines and primary human cells are not internalized upon agonist interaction, but move between cytoplasm and cell membranes by ligand-independent trafficking mechanisms. Even though CB receptors are expressed in many cells of peripheral origin they are not always localized in the cell membrane and in most cancer cell lines the ratios between CB₁ and CB₂ receptor gene and surface expression vary significantly. In contrast, CB receptor cell surface expression in HL60 cells is subject to significant oscillations and CB₂ receptors form oligomers and heterodimers with CB₁ receptors, showing synchronized surface expression, localization and trafficking. We show that hydrogen peroxide and other nonspecific protein tyrosine phosphatase inhibitors (TPIs) such as phenylarsine oxide trigger both CB₂ receptor internalization and externalization, depending on receptor localization. Phorbol ester-mediated internalization of CB receptors can be inhibited via this switch. In primary human immune cells hydrogen peroxide and other TPIs lead to a robust internalization of CB receptors in monocytes and an externalization in T cells. This study describes, for the first time, the dynamic nature of CB receptor trafficking in the context of a biochemical switch, which may have implications for studies on the cell-type specific effects of cannabinoids and our understanding of the regulation of CB receptor cell surface expression

Cellular uptake of metallated cobalamins

Cellular uptake of vitamin B12-cisplatin conjugates was estimated via detection of their metal constituents (Co, Pt, and Re) by inductively coupled plasma mass spectrometry (ICP-MS). Vitamin B12 (cyano-cob(III)alamin) and aquo-cob(III)alamin [Cbl-OH2]+, which differ in the β-axial ligands (CN− and H2O, respectively), were included as control samples. The results indicated that B12 derivatives delivered cisplatin to both cellular cytosol and nuclei with an efficiency of one third compared to the uptake of free cisplatin cis-[PtIICl2(NH3)2]. In addition, uptake of charged B12 derivatives including [Cbl-OH2]+, [{Co}-CN-{cis-PtCl(NH3)2}]+, [{Re}-{Co}-CN-{cis-PtCl(NH3)2}]+, and [{Co}-CN-{trans-Pt(Cyt)(NH3)2}]2+ (Cyt = cytarabin) was high compared to neutral B12, which implied the existence of an additional internalization pathway for charged B12 vitamin analogs. The affinities of the charged B12 derivatives to the B12 transporters HC, IF and TC were similar to that of native vitamin B12

Scale interaction processes during the MAP IOP 12 south föhn event in the Rhine Valley

This paper examines the three-dimensional structure and dynamics of a south föhn flow in the Rhine Valley during its entire life cycle from 29 October 1999 until 31 October 1999. The south föhn event was documented in the framework of the Mesoscale Alpine Programme (MAP). This study investigates the synoptic-scale forcing sources, the dynamical processes driving the circulation of the föhn flow in the complex network of tributaries of the Rhine Valley, and the degree of inhomogeneity on the scale of the FORM (‘FOehn in the Rhine Valley during the MAP’ programme) target area at the bifurcation between the Rhine and Seez Valleys. Several important data sources were used (ground-based Doppler lidar, scintillometers, constant-volume balloons, radiosoundings and surface stations) as well as non-hydrostatic mesoscale simulations.The föhn penetrating the FORM target area on 30 October 1999 is preceded by a nocturnal shallow föhn phase which does not penetrate down to the ground due to katabatic drainage flow from the main transverse (east–west oriented) tributaries in which föhn cannot penetrate. This paper shows the contribution of the main tributaries of the Rhine Valley in directing the föhn flow towards the FORM target area during (i) the shallow föhn phase where the westerly upper-level flow is deflected in the main longitudinal (i.e. north–south oriented) tributaries; (ii) the penetrating föhn phase, where the south/south-westerly upper-level flow is nearly aligned with the longitudinal tributaries. The channelling efficiency of the main longitudinal tributaries of the Rhine Valley (particularly the Domleschg) is higher during the föhn phase when the south-westerly upper-level flow, experiencing mountain-wave-induced downward motion, penetrates these tributaries, than during the shallow föhn.In the FORM target area, the structure of the föhn flow varies on a 1-kilometre horizontal length-scale and the time evolution of the respective location of the cold air pool and the warm föhn air is investigated in detail. Also, flow splitting between the Rhine and Seez Valleys occurs during the entire föhn life cycle, but its vertical extension is maximum during shallow föhn, when lower- and upper-level flows are fully decoupled

Binding and dissociation kinetics of the fluorescent conjugate CBC and Cbi.

<p>Kinetic measurements revealed a subtle difference between rhHC and native HC. (A) Binding kinetics. rhHC was mixed with either CBC or CBC + Cbl/Cbi (all 0.5 µM), pH 7.5, 22°C. Appearance of rhHC·CBC complex was monitored over time according to increasing fluorescence normalized to the maximal amplitude of the signal. The binding rate constants were calculated by computer fitting (solid lines). (B) Dissociation kinetics. rhHC was mixed with either CBC or the non-fluorescent test ligands Cbl and Cbi (all 1 µM, pH 7.5, 22°C, 2 min incubation), whereupon either Cbl (1 µM) or CBC (1 µM) was added. Change in the concentration of rhHC·CBC complex was monitored over time according to the normalized fluorescent response. Dissociation of Cbi was tested in three different preparations of rhHC (all three curves are presented, see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037421#pone-0037421-t002" target="_blank">Table 2</a>). Best fits are shown by solid lines. The dissociation rate constants were calculated from the linear slopes of the produced charts (examples indicated by dashed lines). Alternative linear slopes are shown for rhHC·Cbl dissociation.</p

Absorbance spectra of rhHC-Cbl complexes.

<p>The absorbance spectrum of Cbl bound to rhHC exhibited a normal transition pattern. (A) Spectrum of rhHC (13.8 µM) in either apo-form or saturated with aquo-Cbl (excess removed) with or without sodium azide (2 mM), pH 7.5, 22°C. (B) Spectra of aquo-Cbl (4.48 µM) added stepwise to rhHC (26.3 µM), dilution was corrected, pH 7.5, 22°C. The data were used for calculation of the coefficients of molar absorbance, see main text and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037421#pone-0037421-t001" target="_blank">Table 1</a>.</p

Purification of rhHC.

<p>rhHC purified from approximately 0.5 L HEK293 cell culture supernatant. (A) Sequence alignment of native HC and tagged rhHC. Thrombin cleavage site (<b>bold</b>), Myc-tag (<u>underlined</u>) and 6×His-tag (<i>italic</i>) (B) Coomassie stained SDS/PAGE and (C) Western blot analysis of the different purification steps. Lanes: 1, standards, 2, supernatant of HEK293 cells transfected with rhHC, 3, elution of first Ni²⁺ affinity chromatography (tagged rhHC), 4, peak fraction of first size exclusion chromatography (tagged rhHC), 5, flow-through of second Ni²⁺ affinity chromatography after thrombin cleavage, 6, peak fraction of second size exclusion chromatography, 7, human native HC <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037421#pone.0037421-Nexo2" target="_blank">[9]</a>. (D) and (E) UV-chromatograms at 280 nm of first and second size exclusion chromatography before and after thrombin cleavage on a Superdex 200 column at a flow rate of 48 ml h⁻¹. Elution volumes of Blue Dextran 2000 (V₀) and albumin (V_alb, 67 kDa) are marked with arrows.</p

Internalisation of ¹²⁵I-rhHC into HepG2 cells.

<p>Comparison of rhHC and human native HC with or without pre-treatment with neuraminidase in order to remove terminal sialic acid. (A) SDS/PAGE analysis after treatment with neuraminidase. (B) Internalisation of ¹²⁵I labelled proteins after 0, 1.5, 3 and 6 h incubation time. Standards are indicated with M.</p