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Biomedical subjects

H de Wet

Publications and source records attributed to H de Wet.

7 recordsLinked to original sources
PubMed2003

Glucocorticoids induce rapid up-regulation of mitogen-activated protein kinase phosphatase-1 and dephosphorylation of extracellular signal-regulated kinase and impair proliferation in human and mouse osteoblast cell lines.

A central feature of glucocorticoid (GC)-induced osteoporosis is decreased bone formation, secondary to decreased numbers of functional osteoblasts. We find that ERK activity is essential for serum-induced osteoblast proliferation in vitro because inhibition of MAPK/ERK kinase activity by U0126 completely abolished both serum-induced activation of ERK and proliferation of mouse (MBA-15.4) and human (MG-63) osteoblast cell lines. Dexamethasone (Dex) rapidly (<2 h) inhibits the sustained phase of ERK activation, required for nuclear shift and mitogenesis. This inhibition is reversed by cotreatment with the protein synthesis inhibitor, cycloheximide, and by the GC receptor antagonist, RU486, suggesting a classical transcriptional mechanism. Phosphatase activity was up-regulated by Dex treatment, and inhibition of ERK activity by Dex was also reversed by the protein tyrosine phosphatase inhibitor, vanadate. Coupled with the rapidity of Dex action, this indicates immediate-early gene phosphatase involvement, and we therefore used quantitative, real-time PCR to examine expression profiles of the dual-specificity MAPK phosphatases, MKP-1 and MKP-3. MKP-1, but not MKP-3, mRNA expression was 10-fold up-regulated in both mouse and human osteoblast cell lines within 30 min of Dex treatment and remained elevated for 24 h. MKP-1 protein was also markedly up-regulated following 1-8 h of Dex treatment, and this correlated precisely with dephosphorylation of ERK. Cell proliferation was impaired by Dex treatment, and this was reversed by both RU486 and vanadate. Therefore, MKP-1 up-regulation provides a novel and rapid mechanism, whereby GCs inhibit osteoblast proliferation.

Animals↗
PubMed2002

Modulation by flavonoids of cell multidrug resistance mediated by P-glycoprotein and related ABC transporters.

Cancer cell resistance to chemotherapy is often mediated by overexpression of P-glycoprotein, a plasma membrane ABC (ATP-binding cassette) transporter which extrudes cytotoxic drugs at the expense of ATP hydrolysis. P-glycoprotein (ABCB1, according to the human gene nomenclature committee) consists of two homologous halves each containing a transmembrane domain (TMD) involved in drug binding and efflux, and a cytosolic nucleotide-binding domain (NBD) involved in ATP binding and hydrolysis, with an overall (TMD-NBD)2 domain topology. Homologous ABC multidrug transporters, from the same ABCB family, are found in many species such as Plasmodiumfalciparum and Leishmania spp. protozoa, where they induce resistance to antiparasitic drugs. In yeasts, some ABC transporters involved in resistance to fungicides, such as Saccharomyces cerevisiae Pdr5p and Snq2p, display a different (NBD-TMD)2 domain topology and are classified in another family, ABCG. Much effort has been spent to modulate multidrug resistance in the different species by using specific inhibitors, but generally with little success due to additional cellular targets and/or extrusion of the potential inhibitors. This review shows that due to similarities in function and maybe in three-dimensional organization of the different transporters, common potential modulators have been found. An in vitro 'rational screening' was performed among the large flavonoid family using a four-step procedure: (i) direct binding to purified recombinant cytosolic NBD and/or full-length transporter, (ii) inhibition of ATP hydrolysis and energy-dependent drug interaction with transporter-enriched membranes, (iii) inhibition of cell transporter activity monitored by flow cytometry and (iv) chemosensitization of cell growth. The results indicate that prenylated flavonoids bind with high affinity, and strongly inhibit drug interaction and nucleotide hydrolysis. As such, they constitute promising potential modulators of multidrug resistance.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2001

Sequence requirements of the ATP-binding site within the C-terminal nucleotide-binding domain of mouse P-glycoprotein: structure-activity relationships for flavonoid binding.

Sequence requirements of the ATP-binding site within the C-terminal nucleotide-binding domain (NBD2) of mouse P-glycoprotein were investigated by using two recombinantly expressed soluble proteins of different lengths and photoactive ATP analogues, 8-azidoadenosine triphosphate (8N(3)-ATP) and 2',3',4'-O-(2,4,6-trinitrophenyl)-8-azidoadenosine triphosphate (TNP-8N(3)-ATP). The two proteins, Thr(1044)-Thr(1224) (NBD2(short)) and Lys(1025)-Ser(1276) (NBD2(long)), both incorporated the four consensus sequences of ABC (ATP-binding cassette) transporters, Walker A and B motifs, the Q-loop, and the ABC signature, while differing in N-terminal and C-terminal extensions. Radioactive photolabeling of both proteins was characterized by hyperbolic dependence on nucleotide concentration and high-affinity binding with K(0.5)(8N(3)-ATP) = 36-37 microM and K(0.5)(TNP-8N(3)-ATP) = 0.8-2.6 microM and was maximal at acidic pH. Photolabeling was strongly inhibited by TNP-ATP (K(D) = 0.1-5 microM) and ATP (K(D) = 0.5-2.7 mM). Since flavonoids display bifunctional interactions at the ATP-binding site and a vicinal steroid-interacting hydrophobic sequence [Conseil, G., Baubichon-Cortay, H., Dayan, G., Jault, J.-M., Barron, D., and Di Pietro, A. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 9831-9836], a series of 30 flavonoids from different classes were investigated for structure-activity relationships toward binding to the ATP site, monitored by protection against photolabeling. The 3-OH and aromaticity of conjugated rings A and C appeared important, whereas opening of ring C abolished the binding in all but one case. It can be concluded that the benzopyrone portion of the flavonoids binds at the adenyl site and the phenyl ring B at the ribosyl site. The Walker A and B motifs, intervening sequences, and small segments on both sides are sufficient to constitute the ATP site.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1998

ATPase and multidrug transport activities of the overexpressed yeast ABC protein Yor1p.

The Saccharomyces cerevisiae genome encodes 15 full-size ATP binding cassette transporters (ABC), of which PDR5, SNQ2, and YOR1 are known to be regulated by the transcription factors Pdr1p and Pdr3p (pleiotropic drug resistance). We have identified two new ABC transporter-encoding genes, PDR10 and PDR15, which were up-regulated by the PDR1-3 mutation. These genes, as well as four other ABC transporter-encoding genes, were deleted in order to study the properties of Yor1p. The PDR1-3 gain-of-function mutant was then used to overproduce Yor1p up to 10% of the total plasma membrane proteins. Overexpressed Yor1p was photolabeled by [gamma-32P]2', 3'-O-(2,4,6-trinitrophenyl)-8-azido-ATP (K0.5 = 45 microM) and inhibited by ATP (KD = 0.3 mM) in plasma membranes. Solubilization and partial purification on sucrose gradient allowed to detect significant Yor1p ATP hydrolysis activity (approximately 100 nmol of Pi.min-1.mg-1). This activity was phospholipid-dependent and sensitive to low concentrations of vanadate (I50 = 0.3 microM) and oligomycin (I50 = 8.5 microg/ml). In vivo, we observed a correlation between the amount of Yor1p in the plasma membrane and the level of resistance to oligomycin. We also demonstrated that Yor1p drives an energy-dependent, proton uncoupler-insensitive, cellular extrusion of rhodamine B. Furthermore, cells lacking both Yor1p and Pdr5p (but not Snq2p) showed increased accumulation of the fluorescent derivative of 1-myristoyl-2-[6-(NBD)aminocaproyl]phosphatidylethanolamine. Despite their different topologies, both Yor1p and Pdr5p mediated the ATP-dependent translocation of similar drugs and phospholipids across the yeast cell membrane. Both ABC transporters exhibit ATP hydrolysis in vitro, but Pdr5p ATPase activity is about 15 times higher than that of Yor1p, which may indicate mechanistic or regulatory differences between the two enzymes.

ATP-Binding Cassette Transporters↗
PubMed1988

Simultaneous dislocation of all five carpometacarpal joints.

A case of unique combination of simultaneous dislocation of all five carpometacarpal joints is presented. The mechanism of injury in this case seems to be some manner of punching with a closed fist rather than direct violence. Satisfactory results were achieved using simple principles of manipulation under anesthesia, open reduction, and skeletal fixation, followed by intensive physiotherapy.

Accidents, Traffic↗
PubMed1968

[Safety belts].

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Accident Prevention↗