Biomedical subjects

W J de Grip

Publications and source records attributed to W J de Grip.

At least 19 recordsLinked to original sources

PubMed2003

Analysis of histamine and modeling of ligand-receptor interactions in the histamine H(1) receptor for Magic Angle Spinning NMR studies.

OBJECTIVE AND DESIGN: Investigation of the principles of ligand-receptor interaction in histamine receptors can help to provide a solid foundation for structure-based drug design. Stable isotope labelling of the ligand 'Histamine' has been performed and 1D (13)C CP MAS and 2D Radio Frequency Dipolar Recoupling (RFDR) spectra for the ligand are presented. Hyperfine signals were well spread and did not suffer from any sizable line broadening. The production of H(1) receptor for Magic Angle Spinning NMR studies is currently in progress. TREATMENT: An agonist binding domain is proposed using homology modeling, database searches and mutagenesis data for the H(1) receptor. METHODS: Homology modeling, Database searches for Expressed sequence Tag (ESTs), Magic Angle Spinning Nuclear Magnetic Resonance analysis of the ligand histamine. RESULTS: The three-dimensional receptor model and mutagenesis studies suggest that the amine of the agonist histamine may form an ion pair with the TM III Asp, whereas the imidazole ring of histamine may associate with TM V Asp and Thr. CONCLUSIONS: Homology modeling studies confirms the absence of TM VIII in the H(1) receptor. According to the model the histamine in particular interacts with the transmembrane (TM) regions of the H(1) receptor structure, in particular TM helix III and V. This is in line with recent mutagenesis studies. Database search methods for ESTs have been used for electronic prediction of tissue distribution of H(1) receptor expression. The results indicate that the H(1) expression is highest in heart and skeletal muscle, which may be of importance for drug targeting.

Biophysical Phenomena↗

PubMed2001

Large-scale purification of functional recombinant human aquaporin-2.

The homotetrameric aquaporin-2 (AQP2) water channel is essential for the concentration of urine and of critical importance in diseases with water dysregulation, such as nephrogenic diabetes insipidus, congestive heart failure, liver cirrhosis and pre-eclampsia. The structure of human AQP2 is a prerequisite for understanding its function and for designing specific blockers. To obtain sufficient amounts of AQP2 for structural analyses, we have expressed recombinant his-tagged human AQP2 (HT-AQP2) in the baculovirus/insect cell system. Using the protocols outlined in this study, 0.5 mg of pure HT-AQP2 could be obtained per liter of bioreactor culture. HT-AQP2 had retained its homotetrameric structure and exhibited a single channel water permeability of 0.93+/-0.03x10(-13) cm3/s, similar to that of other AQPs. Thus, the baculovirus/insect cell system allows large-scale expression of functional recombinant human AQP2 that is suitable for structural studies.

Amino Acid Sequence↗

PubMed2000

Determination of a molecular torsional angle in the metarhodopsin-I photointermediate of rhodopsin by double-quantum solid-state NMR.

We present a solid-state NMR study of metarhodopsin-1, the pre-discharge intermediate of the photochemical signal transduction cascade of rhodopsin, which is the 41 kDa integral membrane protein that triggers phototransduction in vertebrate rod cells. The H-C10-C11-H torsional angles of the retinylidene chromophore in bovine rhodopsin and metarhodopsin-I were determined simultaneously in the photo-activated membrane-bound state, using double-quantum heteronuclear local field spectroscopy. The torsional angles were estimated to be [phi] = 160+/-10 degrees for rhodopsin and phi = 180+/-25 degrees for metarhodopsin-I. The result is consistent with current models of the photo-induced conformational transitions in the chromophore, in which the 11-Z retinal ground state is twisted, while the later photointermediates have a planar all-E conformation.

Animals↗

PubMed1999

Eye lens alphaA- and alphaB-crystallin: complex stability versus chaperone-like activity.

The major lens protein alpha-crystallin is composed of two related types of subunits, alphaA- and alphaB-crystallin, of which the former is essentially lens-restricted, while the latter also occurs in various other tissues. With regard to their respective chaperone capacities, it has been reported that homomultimeric alphaA-crystallin complexes perform better in preventing thermal aggregation of proteins, while alphaB-crystallin complexes protect more efficiently against reduction-induced aggregation of proteins. Here, we demonstrate that this seeming discrepancy is solved when the reduction assay is performed at increasing temperatures: above 50 degrees C alphaA- performs better than alphaB-crystallin also in this assay. This inversion in protective capacity might relate to the greater resistance of alphaA-crystallin to heat denaturation. Infrared spectroscopy, however, revealed that this is not due to a higher thermostability of alphaA-crystallin's secondary structure. Also the accessible hydrophobic surfaces do not account for the chaperoning differences of alphaA- and alphaB-crystallin, since regardless of the experimental temperature alphaB-crystallin displays a higher hydrophobicity. It is argued that the greater complex stability of alphaA-crystallin, as evident upon urea denaturation, and the higher chaperone capacity of alphaB-crystallin at physiological temperatures reflect the evolutionary compromise to obtain an optimal functioning of heteromeric alpha-crystallin as a lens protein.

Anilino Naphthalenesulfonates↗

PubMed1999

Retinylidene ligand structure in bovine rhodopsin, metarhodopsin-I, and 10-methylrhodopsin from internuclear distance measurements using 13C-labeling and 1-D rotational resonance MAS NMR.

Rhodopsin is the G-protein coupled photoreceptor that initiates the rod phototransduction cascade in the vertebrate retina. Using specific isotope enrichment and magic angle spinning (MAS) NMR, we examine the spatial structure of the C10-C11=C12-C13-C20 motif in the native retinylidene chromophore, its 10-methyl analogue, and the predischarge photoproduct metarhodopsin-I. For the rhodopsin study 11-Z-[10,20-(13)C(2)]- and 11-Z-[11,20-(13)C(2)]-retinal were synthesized and incorporated into bovine opsin while maintaining a natural lipid environment. The ligand is covalently bound to Lys(296) in the photoreceptor. The C10-C20 and C11-C20 distances were measured using a novel 1-D CP/MAS NMR rotational resonance experimental procedure that was specifically developed for the purpose of these measurements [Verdegem, P. J. E., Helmle, M., Lugtenburg, J., and de Groot, H. J. M. (1997) J. Am. Chem. Soc. 119, 169]. We obtain r(10,20) = 0.304 +/- 0.015 nm and r(11,20) = 0.293 +/- 0.015 nm, which confirms that the retinylidene is 11-Z and shows that the C10-C13 unit is conformationally twisted. The corresponding torsional angle is about 44 degrees as indicated by Car-Parrinello modeling studies. To increase the nonplanarity in the chromophore, 11-Z-[10,20-(13)C(2)]-10-methylretinal and 11-Z-[(10-CH(3)), 13-(13)C(2)]-10-methylretinal were prepared and incorporated in opsin. For the resulting analogue pigment r(10,20) = 0.347 +/- 0.015 nm and r((10)(-)(CH)()3())(,)(13) = 0.314 +/- 0.015 nm were obtained, consistent with a more distorted chromophore. The analogue data are in agreement with the induced fit principle for the interaction of opsin with modified retinal chromophores. Finally, we determined the intraligand distances r(10,20) and r(11,20) also for the photoproduct metarhodopsin-I, which has a relaxed all-E structure. The results (r(10,20) >/= 0.435 nm and r(11,20) = 0.283 +/- 0.015 nm) fully agree with such a relaxed all-E structure, which further validates the 1-D rotational resonance technique for measuring intraligand distances and probing ligand structure. As far as we are aware, these results represent the first highly precise distance determinations in a ligand at the active site of a membrane protein. Overall, the MAS NMR data indicate a tight binding pocket, well defined to bind specifically only one enantiomer out of four possibilities and providing a steric complement to the chromophore in an ultrafast ( approximately 200 fs) isomerization process.

Animals↗

PubMed1999

Solid state 15N NMR evidence for a complex Schiff base counterion in the visual G-protein-coupled receptor rhodopsin.

Using the baculovirus/Sf9 cell expression system, we have incorporated 99% 15N-enriched [alpha,epsilon-15N2]-L-lysine into the rod visual pigment rhodopsin. We have subsequently investigated the protonated Schiff base (pSB) linkage in the [alpha, epsilon-15N2]Lys-rhodopsin with cross-polarization magic angle spinning (CP/MAS) 15N NMR. The Schiff base (SB) 15N in [alpha, epsilon-15N2]Lys-rhodopsin resonates with an isotropic shift sigmaI of 155.9 ppm, relative to 5.6 M 15NH4Cl. This suggests that the SB in rhodopsin is protonated and stabilized by a complex counterion. The 15N shifts of retinal SBs correlate with the energy difference between the ground and excited states and the frequency of maximum visible absorbance, numax, associated with the pi-pi transition of the polyene chromophore. Experimental modeling of the relation between the numax and the size of the counterion with a set of pSBs provides strong evidence that the charged chromophore in rhodopsin is stabilized by a counterion with an estimated effective center-center distance (deff) between the counterion and the pSB of 0.43 +/- 0.01 nm. While selected prokaryotic proteins and complexes have been labeled before, this is the first time to our knowledge that a 15N-labeled eukaryotic membrane protein has been generated in sufficient amount for such NMR investigations.

Animals↗

PubMed1997

Macroscopic orientation of natural and model membranes for structural studies.

One approach for obtaining high-resolution structural and functional information for biomembranes and their proteins is by static solid-state NMR of oriented systems. Here, a general procedure to align fully functional biological membranes containing large membrane proteins (Mr >30,000) is described. The method, based on the isopotential spin-dry ultracentrifugation technique, relies on the centrifugation of membrane fragments onto a support with simultaneous, or subsequent, partial evaporation of the solvent which aids alignment. The quality of orientation, as shown by the mosaic spread of the samples, was monitored by static solid-state 31P NMR for the phospholipids and by 2H NMR for a deuterated retinal in bovine rhodopsin. The generality of this method is demonstrated with three different membranes containing bovine rhodopsin in reconstituted bilayers, natural membranes with the red cell anion exchange transport protein in erythrocytes, band 3, and the nicotinic acetylcholine receptor.

Animals↗

PubMed1997

The effect of ethyldeshydroxy-sparsomycin and cisplatin on the intracellular glutathione level and glutathione S-transferase activity.

Ethyldeshydroxy-sparsomycin (EdSm) is a ribosomal protein synthesis inhibitor which synergistically enhances the antitumor activity of cisplatin against L1210 leukemia in vivo. Because cellular glutathione (GSH) and glutathione S-transferases (GST) are reported to interfere with the antitumor activity of cisplatin, we analyzed the effect of EdSm and cisplatin on GSH and GST activity in selected tumor cells. For this purpose we used three murine leukemia tumors with different sensitivities towards EdSm and cisplatin: L1210-WT, sensitive to both drugs, L1210-Sm, resistant to EdSm, and L1210-CDDP, resistant to cisplatin. No significant differences were detectable between these three cell lines regarding the population doubling time, the cell size, and the cellular level of protein and glutathione. Neither of the resistant L1210 subclones showed P-glycoprotein expression. Drug exposure, however, changed the intracellular dynamics. Exposure to EdSm strongly decreased the amount of cellular protein, decreased the overall GST activity and led to GSH depletion, whereas exposure to cisplatin induced a rise in the amount of protein, in GSH, and in the total GST activity. These effects are dose-dependent and correlate well with the sensitivity of the tumor cells for EdSm or cisplatin. In addition, exposure to EdSm lowered the V(max) of GST in L1210-WT and L1210-Sm; however, in L1210-CDDP both the V(max) and the K(m) were increased. That this was not a direct effect of EdSm on GST was shown in a cell-free system, where EdSm did not influence the GST activity nor could it act as a substrate for GST. Our results suggest that the synergistic combination of EdSm and cisplatin might be explained by EdSm switching off the cellular detoxification mechanism for cisplatin, i.e. by inhibition of de novo synthesis and subsequent depletion of GSH and GST.

Animals↗

PubMed1995

Schedule-dependent enhancement of antitumor activity of ethyldeshydroxy-sparsomycin in combination with classical antineoplastic agents.

The efficacy of the protein synthesis inhibitor ethyldeshydroxy-sparsomycin (EDSM) as a biochemical response modifier of several antitumor agents against L1210 leukemia and B16 melanoma is described. Seven drugs with different intracellular targets were selected for this combination study. Tumor implantation and drug treatment were both i.p., and the time interval between the administration of EDSM and the cytostatic agent was varied. Our results show that in the B16 tumor model EDSM is not able to potentiate any of these drugs, whereas antagonism is seen in combination with doxo-rubicin (DX). In the L1210 tumor model, however, no loss of activity is seen for this specific combination. The effect of the combination of cytosar (Ara-C), 5-fluorouracil (5-FU) or vincristine (VCR) with EDSM in the L1210 model is strongly time interval dependent. Loss of 5-FU antitumor activity is seen when EDSM is given 3 or 24 h after 5-FU; however, no effect is observed when EDSM is given 6 h after 5-FU. Enhancement of the 5-FU activity is not noticed. The VCR activity is potentiated when EDSM is given at least 6 h after VCR administration, which increases the antitumor response from 32 to > 60 days and the percentage survivors from 33 to 83% (p = 0.04). In combination with Ara-C, potentiation of antitumor activity is seen only when EDSM is given 24 h after Ara-C, which increases the antitumor response from 32 to > 55 days and the percentage survivors from 11 to 50% (p = 0.008). No modulatory effects are found when EDSM is combined with carmustine or DX.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Quantitative analysis of residual detergent in proteoliposomes by Fourier transform infrared spectroscopy.

An infrared spectroscopic method has been developed to quantify the residual amount of detergent present in membranes, e.g., after reconstitution of a membrane protein in phospholipids. A detergent-specific band is selected in the ir spectrum and the intensity is ratioed against a phospholipid-specific band (around 1235 cm-1, vas (P = O)). This ratio shows a linear relation with the molar ratio of detergent of phospholipid, down to as low as 1 to 10. The new method is illustrated in two case studies, viz., tris(hydroxyethyl)ammonium cholate in Na+/K(+)-ATPase proteoliposomes and n-dodecyl-beta-D-maltoside in rhodopsin proteoliposomes.

Animals↗

PubMed1994

Rhodopsin's secondary structure revisited: assignment of structural elements.

FT-IR spectroscopy has been applied to study the secondary structure of rhodopsin in dehydrated films of bovine rod photoreceptor membranes. Curve fitting analysis of the amide I band around 1658 cm-1 compares well with data obtained from samples in the hydrated state. Repeating this analysis on samples, treated with proteinase K or thermolysin, secondary structural elements at the cytoplasmic side of the photoreceptor membrane can be located. We present evidence for the location of a beta-sheet and a beta-turn near the lipid anchor in the C-terminal region of the protein, and for an alpha-helical structure in the third cytoplasmic loop.

Endopeptidase K↗

PubMed1994

Concentration and sequence dependent synergism of ethyldeshydroxy-sparsomycin in combination with antitumor agents.

The modulating effect of ethyldeshydroxy-sparsomycin (EDSM), an inhibitor of ribosomal protein synthesis, on cytostatic agents was studied on cultured B16 melanoma cells using the microculture tetrazolium test (MTT). The data were analyzed for true synergism using the combination index and the median effect principle. The extent of cytotoxic drug interaction was influenced by the duration of drug exposure, the dose ratio, as well as the treatment schedule. When drug ratios were used, synergism was observed upon pre- and post-treatment in combination with cisplatin, cytosine arabinoside (Ara-C), methotrexate (MTX) and 5-fluorouracil (5-FU). The combination of a fixed dose of EDSM was synergistic with cisplatin, Ara-C, vincristine (VCR) and MTX, in the order MTX > Ara-C > VCR > cisplatin, while the combinations with doxorubicin, 5-FU and etoposide (VP-16) were shown to be antagonistic. These results suggest that only certain drugs and treatment schedules might be worthwhile for combination studies with EDSM in vivo and indicate a role for EDSM as modifier of antitumor responses in cancer chemotherapy.

Animals↗

PubMed1993

Rod-opsin immunoreaction in the pineal organ of the pigmented mouse does not indicate the presence of a functional photopigment.

The aim of the present study was to characterize the rod-opsin immunoreaction in the mammalian pineal organ. Pigmented mice (strain C57BL) were selected as the animal model. Immunocytochemical investigations involving the use of highly specific polyclonal and monoclonal antibodies against bovine rod-opsin (the apoprotein of the photopigment rhodopsin) showed that approximately 25% of all pinealocytes were rod-opsin immunoreactive. Immunoblotting techniques revealed three protein bands of approximately 40, 75, and 110 kDa; these were detected by the monoclonal antibody and the polyclonal antiserum in retinal and pineal extracts. These protein bands presumably represented the monomeric, dimeric and trimeric forms of rod-opsin. The amount of rod-opsin in retina and pineal organ was quantified by means of an enzyme-linked immunosorbent assay. This yielded 570 +/- 30 pmoles rod-opsin per eye and 0.3 +/- 0.05 pmoles rod-opsin per pineal organ. High pressure liquid chromatography analysis of whole eye extracts demonstrated the chromophoric group of the photopigment rhodopsin, 11-cis retinal, and its isomer, all-trans-retinal. A shift from 11-cis retinal to all-trans-retinal was found upon light adaptation. No retinals were detected in the pineal organ. Autoradiographic investigations showed that 3H-retinol, intraperitoneally injected into the animals, was incorporated into the outer and inner segments of retinal photoreceptors, but not into the pineal organ. It is concluded that the mouse pineal organ contains the authentic apoprotein of rhodopsin but that it lacks retinal derivatives as essential components of all known vertebrate photopigments.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Erythrocyte band 3-like protein immunoreactivity in the human brain cortex.

The immunoreactivity of erythrocyte band 3 (B3-IR)-related protein was estimated on cortex biopsies from the brains of 33 patients varying in age from 14-week-old fetus until 67 years of life. B3-IR was not a feature of embryonic brains. A positive reaction was restricted to neurons. It appeared at early postnatal life, increased sharply until 9 years, and than stayed approximately stable between 17 and 67 years of age. The results indicate that there is a positive relation between the amount of neuronal band 3-like protein and the stage of human brain development.

Adolescent↗

PubMed1991

Photoreceptor differentiation in cerebellar medulloblastoma: evidence for a functional photopigment and authentic S-antigen (arrestin).

The aim of the present study was to evaluate the putative photoreceptor differentiation found in certain cerebellar medulloblastomas. The analyses were focussed on S-antigen, rod-opsin (the apoprotein of the visual pigment rhodopsin) and 11-cis retinal (the prosthetic group of rhodopsin). Fresh frozen and paraffin-embedded biopsy specimens of three medulloblastomas were investigated by means of immunocytochemistry, enzyme-linked immunosorbent assay (ELISA), high-pressure liquid chromatography (HPLC), and immunoblotting. As shown in paraffin sections, one out of the three tumors (tumor A) contained S-antigen- and rod-opsin-immunoreactive tumor cells. The immunoblotting technique revealed in this tumor a single protein band of approximately 48-50 kDa that reacted with the S-antigen antibody and three protein bands of approximately 40, 75 and 110 kDa recognized by the rod-opsin antibody. These bands could not be detected in the two remaining tumors (tumor B and C). The rod-opsin content of tumor A was quantified by the ELISA; 11.7 pmol rod-opsin were calculated for the biopsy. The HPLC demonstrated the presence of 11-cis- and all-trans-retinal in tumor A, but not in tumors B and C. Furthermore, it was shown that 11-cis-retinal was converted to all-trans-retinal upon illumination of the tumor extract. The ratio between 11-cis- and all-trans-retinal was approximately 1:1 before illumination and 3:5 after illumination. A total of 2-3 pmol of retinal was found in the biopsy of tumor A. In addition all-trans-retinol was present in this tumor. The results indicate that certain medulloblastomas express a functional photopigment and S-antigen, another protein of the phototransduction cascade. They strongly support the concept that medulloblastoma cells may differentiate along the photoreceptor cell lineage.

Antigens↗

PubMed1991

Uptake and isomerization of all-trans retinol by isolated bovine retinal pigment epithelial cells: further clues to the visual cycle.

The site and substrate for all-trans to 11-cis isomerization in the visual cycle have remained obscure for several decades. Only recently studies on a subcellular level have begun to shed some light on these phenomena. We have addressed this system on a cellular level by utilizing intact isolated bovine retinal pigment epithelial cells, maintained during short-term incubation in vitro. Supplementation with labeled all-trans retinol incorporated in a lipid vesicle carrier, in a range of 1-6 nmol per 10(6) cells, resulted in a rapid uptake of retinol. The majority of the internalized retinol was processed prior to mixing with endogenous retinoid pools and most of it was converted into all-trans retinylester. Up to 10% of the incorporated label was isomerized yielding 11-cis retinol, 11-cis retinaldehyde and 11-cis retinylester. The kinetics of the 11-cis retinoid formation indicated that 11-cis retinol is the first isomerization product. The level of 11-cis retinol apparently 'triggered' further processing into other 11-cis retinoids. An updated model with discussion topics is presented for the retinoid pathway relevant to the visual cycle.

Animals↗

PubMed1991

Alzheimer's disease and cellular aging: membrane-related events as clues to primary mechanisms.

A fast-increasing number of data indicate that Alzheimer's disease is a systemic disease, not restricted to the central nervous system. The nature of Alzheimer-related changes in peripheral cells confirms and emphasizes indications from biochemical and morphological studies in brain for the involvement of cellular membranes in this disease. Analysis of membrane changes in erythrocytes from Alzheimer patients suggests that the normal aging process of these cells is disturbed. These conclusions may form the basis for further studies leading to a better insight into the etiology of Alzheimer's disease and to the development of diagnostic tools.

Aged↗

PubMed1990

10,20-Methanorhodopsins: (7E,9E,13E)-10,20-methanorhodopsin and (7E,9Z,13Z)-10,20-methanorhodopsin. 11-cis-locked rhodopsin analog pigments with unusual thermal and photo-stability.

Synthesis of the retinal analog, 10,20-methanoretinal (R6), where the 11Z conformation is locked in a six-membered ring, yielded four stereoisomers (7E,9E,13E, 7E,9E,13Z, 7E,9Z,13E and 7E,9Z,13Z). These four isomers were separated by straight-phase isocratic HPLC and identified by 1H-NMR and NOE analysis. All isomers smoothly recombined with bovine opsin at a relatively high rate (5-10% of that of the natural chromophore 11Z-retinal). The corresponding 13E and 13Z isomers yielded identical analog pigments, probably due to rapid thermal isomerization around the C13 = C14 double bond. The (7E,9E)-isomers produced a pigment with maximal absorbance at 510 nm, while the pigment produced from the (7E,9Z)-isomers had maximal absorbance at 494 nm. Based upon kinetic considerations, the chromophore structure in the 510-nm-absorbing pigment should be (7E,9E,13E), i.e. equivalent to 11Z-retinal and rhodopsin, while the chromophore structure in the 494-nm-absorbing pigment should be (7E,9Z,13Z), i.e. equivalent to (9Z,11Z,13Z)-rhodopsin, an isorhodopsin analog. In analogy to the 11-cis-locked rhodopsin analogs Rh5 and Rh7, the 510-nm-absorbing pigment, (7E,9E,13E)-10,20-methanorhodopsin, was dubbed Rh6 and the 494-nm-absorbing pigment. (7E,9Z,13Z)-10,20-methanorhodopsin, was dubbed Iso6. The opsin shift of Rh6 (2660 cm-1) is practically identical to that of rhodopsin itself (2650 cm-1). Rh6 and Iso6 are nearly as stable as rhodopsin towards hydroxylamine and solubilization in detergent solution and could be easily purified and reconstituted into proteoliposomes by established procedures. Due to the 11-cis-lock, Rh6 is much less photolabile (bleaching rate less than 1%) than rhodopsin, but it is not completely photostable, probably since photoisomerization around the C7 = C8, C9 = C10 and C13 = C14 bonds is allowed. Illumination of either Rh6 or Iso6 does not generate the common photointermediates but instead produces a complex pattern of photochemical transitions, which during continuous illumination leads to the same final steady state, absorbing at 498 nm. This process is accompanied by a slow but steady loss of pigment, probably due to hydrolytic release of chromophore, which is markedly accelerated in the presence of hydroxylamine. In a physiological assay (light-dependent activation of rod cGMP phosphodiesterase) Rh6 is only marginally active and this probably reflects conformational changes accompanying the above-mentioned photochemical transitions. This supports the concept that normal rhodopsin-based phototransduction requires 11Z to all-E isomerization.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗