Michel Verhas

Surface plasmon resonance: principles, methods and applications in biomedical sciences

Surface plasmon resonance (SPR) is a phenomenon occuring at metal surfaces (typically gold and silver) when an incident light beam strikes the surface at a particular angle. Depending on the thickness of a molecular layer at the metal surface, the SPR phenomenon results in a graded reduction in intensity of the reflected light. Biomedical applications take advantage of the exquisite sensitivity of SPR to the refractive index of the medium next to the metal surface, which makes it possible to measure accurately the adsorption of molecules on the metal surface and their eventual interactions with specific ligands. The last ten years have seen a tremendous development of SPR use in biomedical applications. The technique is applied not only to the measurement in real-time of the kinetics of ligand-receptor interactions and to the screening of lead compounds in the pharmaceutical industry, but also to the measurement of DNA hybridization, enzyme-substrate interactions, in polyclonal antibody characterization, epitope mapping, protein conformation studies and label-free immunoassays. Conventional SPR is applied in specialized biosensing instruments. These instruments use expensive sensor chips of limited reuse capacity and require complex chemistry for ligand or protein immobilization. Our laboratory has successfully applied SPR with colloidal gold particles in buffered solution. This application offers many advantages over conventional SPR. The support is cheap, easily synthesized, and can be coated with various proteins or protein-ligand complexes by charge adsorption. With colloidal gold, the SPR phenomenon can be monitored in any UV-vis spectrophotometer. For high-throughput applications, we have adapted the technology in an automated clinical chemistry analyzer. This simple technology finds application in label-free quantitative immunoassay techniques for proteins and small analytes, in conformational studies with proteins as well as in the real-time association-dissociation measurements of receptor-ligand interactions, for high-throughput screening and lead optimization.

High-throughput screening using the surface plasmon resonance effect of colloidal gold nanoparticles

We propose a high-throughput screening method which involves colloidal gold nanoparticles sensitized with the binding protein. Upon interaction with a specific ligand (a polypeptide or a small organic molecule), the surface plasmon resonance absorbance peak of the colloidal gold reagent shifts toward longer wavelengths due to the change in refractive index at the particle surface caused by changes in mass. The shift is proportional to the dose of ligand involved for a fixed amount of binding protein and occurs according to the kinetics of interaction. We applied this property to the analysis of association and dissociation of ligand–binding protein interactions in a small random access clinical chemistry analyzer. The instrument measures the changes in A600 nm over a period of 20 min for each sample. Due to the high degree of automation, the instrument throughput amounts to 144 samples an hour and can be run during 24 h a day in a walk-away mode. When connected to a computer for data handling, a single instrument can consequently handle over 3000 samples a day. Higher throughput instruments are available which can handle as much as ten times more samples. We validated the technique by comparing the affinity constants (range 103−1012 mol−1) calculated for 30 pairs of ligand-protein interactions at different ligand doses with those obtained from other methods, including the BIAcore (slope 0.84; coefficient of correlation r = 0.82).

Lack of evolution of the cerebral blood flow during clinical recovery of a stroke.

Cerebral blood flow and clinical parameters were studied in 30 stroke patients at 15th, 30th, 60th, 90th days after the cerebral insult (Xenon 133 inhalation method). The clinical improvement was not accompanied by a progressive normalization of the CBF at rest. No relationship was found between the clinical data and the CBF values; either on the affected hemisphere or on the contralateral one. It is concluded that measurement of the CBF at rest has no predictive value as regards further clinical evolution.

Advances in High-Throughput Screening: Biomolecular Interaction Monitoring in Real-Time with Colloidal Metal Nanoparticles

The post-genomic era is revolutionizing the drug discovery process. The new challenges in the identification of therapeutic targets require efficient technological tools in order to be properly addressed. Label-free detection systems use proteins or ligands coupled to materials of which the physical properties are measurably modified upon specific interactions. Among the label-free systems currently available, the use of metal nanocolloids offers enhanced throughput and flexibility for real-time biomolecular recognition monitoring at a reasonable cost.

Magnesium bioavailability from mineral water. A study in adult men.

Objective: To assess magnesium enteral absorption from a magnesium-rich mineral water.Design: Experimental study.Setting: Department of Nuclear Medicine, Brugmann Hospital, Brussels, Belgium.Subjects: Ten healthy male volunteers in the age range 25–42 y.Intervention: Each subject completed two sessions in a random order. At one session, they received an oral load of 300 ml of water (containing 1.2 mmol Mg), traced with 28Mg, and at the other session they received an intravenous injection of 28Mg, in order to take into account the metabolism of endogenous magnesium. The dietary consumption was further noted on a weekly diary.Results: The mean bioavailability was 59.1% (s.d.±13.6). Magnesium absorption and age were significantly inversely correlated (r=−0.68, P=0.035).Conclusion: Magnesium-rich mineral water is a reliable source of magnesium. Our observation of decreased magnesium absorption with age deserves further investigations.Sponsorship: The study was sponsored by SEV, Bourg la Reine, France.

Demineralization and pathological physiology of the skeleton in paraplegic rats

SummaryThe mechanism of bone loss after a spinal cord section with paraplegia is still largely unknown. The purpose of this study was to investigate in paraplegic rats the rate and distribution of bone loss, changes in bone calcium metabolism, and bone blood flow. Female Sprague-Dawley rats aged 100–120 days were rendered paraplegic by sectioning the spinal cord at the 11th dorsal vertebra.By comparison with their sham-operated controls (SO controls), we found a diminished bone calcium content in the tibia and femur (paralyzed region) and in the humerus (supralesional region) of the paraplegic animals. In the femur bone calcium loss was present within a week and at 12 weeks had reached 22%; in the tibia it started at about 2 weeks and at 12 weeks had reached 15%; in the humerus it started at about 5 weeks and reached 7.5%. It was not uniform and was greatest in the metaphyseal-epiphyseal regions on either side of the femorotibial joint. The blood flow in femur and tibia was measured by the technique of arteriolar blockade of 15 µm microspheres. It was continuously higher in the paraplegic animals than in the SO controls, from 1 to 12 weeks. In both groups, paraplegics and controls, the bone blood flow was unequally distributed in the two bones; it was greatest in the same metaphyseal-epiphyseal regions contiguous to the femorotibial joint.The 72 h45Ca clearance by the femur and tibia was lower in the paraplegic animals, indicating that bone deposition had slowed down and perhaps that resorption had occurred.The fact that the site of maximum bone calcium loss and the site where the blood flow was greatest are the same suggests a close relationship between the increased blood flow and demineralization, whether it be causal or not.

Pathogenesis of aphasia in deep-seated lesions: likely role of cortical diaschisis.

In order to study the pathophysiology of language disorders due to deep-seated left-hemisphere lesions not involving the cortex, a population of 43 right-handed stroke patients (29 aphasic) presenting with such lesions was studied clinically and by regional cerebral blood flow measurements (two-dimensional xenon-133 inhalation method). Most of the patients were studied sequentially between the 1st and 3rd months after stroke. Cortical diaschisis did not account for the whole clinical picture: the remote effect on the cortex could explain the occurrence of aphasia and its severity but not the type of aphasia. Furthermore, the clinical improvement observed in most cases was not accompanied by the disappearance of the diaschisis.

Influence of patient's weight on dual-photon absorptiometry and dual-energy X-ray absorptiometry measurements of bone mineral density

Lumbar spine bone mineral density (BMD) was measured by dual-energy X-ray absorptiometry (DXA) (Hologic QDR 1000) and by153Gd dual-photon absorptiometry (DPA) (Novo Lab 22a) in 120 postmenopausal women. Though a high correlation existed between the two techniques, the ratio between DXA and DPA values was not constant. Using DXA we observed a higher dependence of BMD on weight than in the DPA measurements. To investigate the different behaviour of DXA and DPA machines with weight, we analysed the effects of increasing thickness of soft tissue equivalents on the BMD of the Hologic spine phantom and on the BMD equivalent of an aluminium standard tube. Increasing tissue-equivalent thickness caused the phantom BMD measured by DPA to decrease significantly but had not effect on the DXA measurements. The different behaviour of DPA and DXA equipment with regard to the phantoms could account for the differences observed in the relations between BMD and weight in the patients. Using multiple regression we studied the influence of weight and body mass index on the relation between BMD measured by the two techniques. The introduction of either of these variables into the regression resulted in an improvement of the prediction of the DXA values from the DPA values. However, the residual standard error of the estimate was still higher than the combined precision errors of the two methods, so that no simple relation allows a conversion of BMDDPA into BMDDXA. Our results confirm that BMD is positively correlated with weight in postmenopausal women; the influence of weight on BMD is blunted when the Novo Lab 22a DPA machine is used for measuring bone mineral.

Prognostic value of computed tomography in aphasic stroke patients

In order to evaluate the prognostic value of CT scan in aphasic stroke patients, a prospective study was performed during the first 3 months of the disease. The severity of the language disorders was assessed by means of a quantitative method. In cortico-subcortical lesions, the verbal expression recovery rate and the final verbal expression and comprehension status depended on the infarct size. No relation was observed between the latter and the verbal comprehension recovery rate. In deep-seated lesions, computed tomography provided no information concerning the prognosis.

Effects and interactions of 17β-estradiol, T3 and 1,25(OH)2D3 on cultured osteoblasts from mature rats

Osteoporosis being frequently associated with hyperthyroidism and, mostly after menopause, with deficiency in estrogens, we tried to elucidate the interactions of estrogens and triiodothyronine (T3) with calcitriol by using cultured osteoblast-like cells obtained from mature rat bone. The tested parameters included [3H]thymidine incorporation, evaluation of the alkaline phosphatase activity of the cell layer and osteocalcin production in the culture medium. At physiological concentrations, 17 beta-estradiol and T3 stimulated alkaline phosphatase activity, did not enhance osteocalcin production and slightly inhibited [3H]thymidine incorporation. At higher concentrations, 17 beta-estradiol decreased the alkaline phosphatase and osteocalcin response to calcitriol whereas T3, although decreasing alkaline phosphatase activity, markedly increased the osteocalcin secretion elicited by calcitriol. These observations emphasize the complex physiology of osteoblasts and confirm different behaviors of alkaline phosphatase and of osteocalcin as markers of bone turnover.