Microstructure analysis and mechanical properties by instrumented indentation of Charonia Lampas Lampas shell.

Scanning electron microscopy, X-ray diffraction and Fourier transformed infrared spectroscopy have been used to characterize the microstructure and instrumented microindentation for the determination of the mechanical properties of Charonia Lampas Lampas shell. Both elastic modulus and hardness are found to be dependent on the texture of the three distinct layers. From the analysis of load-depth curves, the shell exhibits small viscoelastic behaviour at low indentation loads and mainly elastoplastic behaviour at higher loads. These phenomena were attributed to the influence of the organic matter present in the shell. Both elastic modulus and hardness are found to be load-dependent in each layer in relation to their microstructure and, accordingly, to the anisotropy of the predominant mineral part. At a macroscopic scale, this tendency is explained by using a rule of mixture and jointly by the anisotropy of the aragonite. The Bull and Page model is subsequently applied to the hardness variation in order to compute the macrohardness which is the characteristic hardness number of a material and the hardness parameter related to the indentation size effect. This model describes well the experimental results for the relative higher depths, and deviates for the small depths due to the effect of the viscoelastic behaviour which then requires a more appropriate model to describe this phenomenon.

Biocompatibility of sol-gel hydroxyapatite-titania composite and bilayer coatings.

Titania-Hydroxyapatite (TiO2/HAP) reinforced coatings are proposed to enhance the bioactivity and corrosion resistance of 316L stainless steel (316L SS). Herein, spin- and dip-coating sol-gel processes were investigated to construct two kinds of coatings: TiO2/HAP composite and TiO2/HAP bilayer. Physicochemical characterization highlighted the bioactivity response of the TiO2/HAP composite once incubated in physiological conditions for 7days whereas the TiO2/HAP bilayer showed instability and dissolution. Biological analysis revealed a failure in human stem cells adhesion on TiO2/HAP bilayer whereas on TiO2/HAP composite the presence of polygonal shaped cells, possessing good behaviour attested a good biocompatibility of the composite coating. Finally, TiO2/HAP composite with hardness up to 0.6GPa and elastic modulus up to 18GPa, showed an increased corrosion resistance of 316L SS. In conclusion, the user-friendly sol-gel processes led to bioactive TiO2/HAP composite buildup suitable for biomedical applications.

Indentation size effect of cortical bones submitted to different soft tissue removals.

Properties of elasticity, hardness and viscosity are determined for the study of the visco-elastoplastic behavior of bones. The mechanical properties are compared in two upright sections of the bone due to their anisotropy. Besides, influence of hydration treatments leading to structural modifications of collagen and ground substance contents of bones on the mechanical properties is studied on a femoral cortical bovine bone. The treatments applied to the bone are used by forensic anthropologists to remove the soft tissue and modifying the hydration degree coupled to the collagen content. From instrumented indentation experiments, the hardness is characterized by the macrohardness and a hardness length-scale factor stating the hardness-load dependence. The elastic modulus results from the application of the methodology of Oliver and Pharr (1992). The coefficient of viscosity is deduced from a rheological model representing the indenter time-displacement observed under the application of a constant load. As a result, all the mechanical properties are found to be lower in the transverse section in an extent depending on the hydration treatment, i.e. the different values are located between 5% and 25% for the hardness around 0.5GPa, between 25% and 40% for the elastic modulus around 20GPa and between 2% and 35% for the coefficient of viscosity around 60GPa.s. Unexpectedly, the elastic modulus to coefficient of viscosity ratio is found to be independent on the hydration treatment.

Characterization of SR 121463A, a highly potent and selective, orally active vasopressin V2 receptor antagonist.

SR 121463A, a potent and selective, orally active, nonpeptide vasopressin V2 receptor antagonist, has been characterized in several in vitro and in vivo models. This compound displayed highly competitive and selective affinity for V2 receptors in rat, bovine and human kidney (0.6 < or = Ki [nM] < or = 4.1). In this latter preparation, SR 121463A potently antagonized arginine vasopressin (AVP)-stimulated adenylyl cyclase activity (Ki = 0.26+/-0.04 nM) without any intrinsic agonistic effect. In autoradiographic experiments performed in rat kidney sections, SR 121463A displaced [3H]AVP labeling especially in the medullo-papillary region and confirmed that it is a suitable tool for mapping V2 receptors. In comparison, the nonpeptide V2 antagonist, OPC-31260, showed much lower affinity for animal and human renal V2 receptors and lower efficacy to inhibit vasopressin-stimulated adenylyl cyclase (Ki in the 10 nanomolar range). Moreover, OPC-31260 exhibited a poor V2 selectivity profile and can be considered as a V2/V1a ligand. In normally hydrated conscious rats, SR 121463A induced powerful aquaresis after intravenous (0.003-0.3 mg/kg) or oral (0.03-10 mg/kg) administration. The effect was dose-dependent and lasted about 6 hours at the dose of 3 mg/kg p.o. OPC-31260 had a similar aquaretic profile but with markedly lower oral efficacy. The action of SR 121463A was purely aquaretic with no changes in urine Na+ and K+ excretions unlike that of known diuretic agents such as furosemide or hydrochlorothiazide. In addition, no antidiuretic properties have been detected with SR 121463A in vasopressin-deficient Brattleboro rats. Thus, SR 121463A is the most potent and selective, orally active V2 antagonist yet described and could be a powerful tool for exploring V2 receptors and the therapeutical usefulness of V2 blocker aquaretic agents in water-retaining diseases.

Angiotensin receptors from rat liver, brain and pituitary gland. Expression of two subtypes in Xenopus oocytes.

Xenopus laevis oocytes were used to express angiotensin receptors encoded by mRNAs extracted from rat liver, adenohypophysis and brain. Groups of ten mRNA-injected oocytes were loaded with 45Ca2+ and the responsiveness to angiotensin II (A II) and related molecules tested by monitoring 45Ca2+ outflux. A II and angiotensin III (A III) induced a marked and transient increase in 45Ca2+ outflux from mRNA, but not from control, water-injected, oocytes. The increase over basal value of 45Ca2+ outflux during a 5 min application period of A II or A III was used as a response index. Observed responses were of high magnitude, reproducible and dose-dependent. For these reasons, mRNA-injected oocytes constitute a valuable system for investigating the pharmacological properties of angiotensin receptors from tissues of different origin under experimental conditions which eliminate tissue-specific interference which might be encountered in classical binding studies on acellular preparations. We demonstrate a fairly good parallelism between the relative potencies of A I, A II and A III in eliciting an increase in 45Ca2+ outflux from liver and adenohypophyseal mRNA-injected oocytes and the relative affinities of these peptides for binding to liver or adenohypophyseal membranes (A II greater than A III much greater than A I). The predominant receptor subtype expressed by brain mRNA discriminated very poorly between A II and A III, whereas angiotensin receptors expressed by liver or adenohypophyseal mRNA discriminated between AII and AIII very efficiently.

Oxytocin receptors from LLC-PK1 cells: expression in Xenopus oocytes.

Two selective radioligands for oxytocin receptors, [3H]-[4-threonine,7-glycine]oxytocin [( 3H]-[Thr4,Gly7]OT) and 125I-[1-(beta-mercapto-beta,beta-cyclopentamethylenepropionic acid), 2-(O-methyl)tyrosine, 4-threonine, 8-ornithine, 9-tyrosine amide]-oxytocin (125I-OTA), were used to characterize oxytocin receptors from two pig kidney-derived cell lines, LLC-PK1 and LLC-PK1L. [3H]-[Thr4,Gly7]OT and 125I-OTA bind with high affinity (mean Kd values of 14 and 0.06 nM, respectively) to the same population of sites on LLC-PK1 cell membranes [maximum binding (Bmax) of 100 fmol/mg membrane protein]. These sites had the expected ligand selectivity of oxytocin receptors. [3H]-[Thr4,Gly7]OT and 125I-OTA binding sites could be distinguished from V2 vasopressin receptors present on LLC-PK1 and LLC-PK1L cells on the basis of clearly different maximal capacities and ligand selectivities, different sensitivities to insulin and serum, and absence of heterologous downregulation. Oxytocin receptors from LLC-PK1 cells have no functional relationship with adenylate cyclase. [Thr4,Gly7]OT affected neither the basal adenosine 3',5'-cyclic monophosphate (cAMP) content nor the vasopressin-induced cAMP accumulation by LLC-PK1 cells. Xenopus laevis oocytes injected with LLC-PK1 cell mRNA responded to [Thr4,Gly7]OT by an increase in 45Ca2+ outflux; this effect is antagonized by a highly selective oxytocin antagonist.

Evidence of two steps in the homologous desensitization of vasopressin-sensitive phospholipase C in WRK1 cells. Uncoupling and loss of vasopressin receptors.

The exposure of WRK1 cells to arginine vasopressin (AVP), lysine vasopressin, or oxytocin for 18 h at 37 degrees C induced a homologous desensitization of the vasopressin- (VP) receptors. Dose-response curves of [3H]lysine vasopressin binding to control and desensitized WRK1 cells revealed a decrease in the maximal number of binding sites without any modification of its affinity (Kd values = 4.40 +/- 0.76 nM and 4.65 +/- 0.78 nM for control and desensitized conditions, respectively). The phenomenon was time- and dose-dependent. It was directly related to receptor occupancy, since the concentration of VP analogues leading to a half-maximal occupancy of VP receptors was closely related to the concentration of the corresponding analogue leading to a half-maximal decrease in VP-binding sites. It was also agonist-specific, since the V1 vasopressin antagonist desGly9d(CH2)5[D-Tyr(Et)2]VAVP was unable to affect the number of receptors. These desensitization processes were completely inhibited when the functional coated pits present in WRK1 cells were suppressed, indicating that the loss of VP-binding sites was related to receptor internalization. The exposure of WRK1 cells to a vasopressin agonist for 18 h also led to an inhibition of the vasopressin-sensitive phospholipase C activity. It was time- and agonist-dose-dependent, and occurred without any detectable changes in apparent affinity values (1.40 +/- 0.04 and 1.90 +/- 0.36 nM for control and desensitized cells, respectively). Control experiments showed that these inhibitions could not have been caused by a decrease in the labeling of inositol lipids. It is likely that they were mainly due to receptor internalization since (i) the hormonal treatment did not modify the basal level of phospholipase C; (ii) the maximal loss of VP-binding site was similar to the maximal inhibition of VP-stimulated IP accumulation; (iii) the recoveries of both VP-binding sites and VP-sensitive phospholipase C activity followed exactly the same time course (t1/2 = 4 h). In addition to this homologous desensitization of VP-sensitive phospholipase C activity, AVP also induced heterologous desensitization of bradykinin-sensitive phospholipase C activity. However, this effect was relatively weak (maximal inhibition 17 +/- 3%). The time course of VP-sensitive phospholipase C desensitization was more rapid than that of VP-receptors, indicating that desensitization involved at least two distinct steps, a rapid uncoupling step, and a later loss of vasopressin receptors.(ABSTRACT TRUNCATED AT 400 WORDS)

A comparative study of plasma vasopressin levels and V1 and V2 vasopressin receptor properties in congenital hypothyroid rat under thyroxine or vasopressin therapy.

The effects of propylthiouracil (PTU) treatment on the plasma vasopressin level, on the number of hepatic (V1) or renal (V2) vasopressin receptors and on the hormone-sensitive adenylate cyclase activity in the kidney of developing rats were studied in parallel. In addition, we investigated the corrective effects of thyroxine therapy on the plasma vasopressin level and parameters related to the liver, and the effects of vasopressin therapy on the parameters related to the kidney. As already reported in the case of the number of V2 receptors and adenylate cyclase activity in the kidney, the deficient plasma vasopressin level in hypothyroid rats was completely corrected by two daily physiological doses of thyroxine given from birth to the age of sacrifice (1 month). Unlike the V1 receptors, the V2 receptors are known to be highly dependent on their specific circulating ligand. Since, first of all, the deficit was similar in the numbers of V1 and V2 receptors in hypothyroid rats, and, secondly, the treatment of hypothyroid rats by two daily physiological doses of long lasting vasopressin was found ineffective to recover the deficit in the number of V2 receptors, it can be concluded that thyroid deficiency directly alters vasopressin receptor biosynthesis in both liver and kidney, instead of acting via the depressed plasma vasopressin level.

Vasopressin, angiotensin and adrenergic receptors of rat liver Golgi fractions--molecular weight of the angiotensin-receptor irreversible complex after in vitro and in vivo labelling.

The binding of vasopressin, angiotensin II and prazosin (alpha 1-adrenergic antagonist) to purified heavy (GH) and (intermediate + light) (GI + L) rat liver Golgi fractions was studied. The three types of ligands showed a saturable and specific binding in Golgi fractions; the maximal specific binding of [3H]vasopressin, [3H]prazosin and [125I]Sar-N3-Phe-angiotensin II was respectively 5-10%, 20-30% and 30-40% of that detected in purified plasma membranes. The apparent binding affinities of the three ligands were the same whether determined in Golgi fractions or plasma membranes. The presence of vasopressin, alpha 1-adrenergic and angiotensin receptors in very different proportions, as compared to the amount of receptor detected in plasma membranes, in GH and GI + L Golgi fractions was not compatible with the idea that a plasma membrane impurity accounted for the detection of receptor in the purified intracellular particulate fractions. In vivo injection of [125I]Sar-N3-Phe-angiotensin II resulted in a receptor-mediated endocytosis of the iodo-angiotensin analog into the GH and GI + L Golgi fractions. The apparent molecular weight of the irreversible complex, [125I]angiotensin-receptor, was estimated in subcellular fractions using SDS-PAGE electrophoresis. This value was identical after either in vivo or in vitro labelling (MW = 63,000) and was indistinguishable from the molecular weight of the irreversible hormone receptor complex present in the plasma membranes.

Comparative study of the developmental patterns of vasopressin, glucagon, angiotensin II, and alpha 1-adrenergic receptors in the liver of developing and adult hypothyroid rats.

The effects of propylthiouracil (PTU) treatment on vasopressin, angiotensin II, glucagon and alpha 1-adrenergic receptors in both developing and adult rats were studied in liver membrane preparations by measuring the binding of the following ligands: [3H][8-lysine]vasopressin, [3H]Sar-angiotensin II, [125I]glucagon and [3H]prazosin, and in the case of glucagon, by measuring adenylate cyclase activation. Whatever the ligand used, in young as well as in adult animals, PTU treatment led to a similar reduction (about 50%) in the maximal number of binding sites (Bmax), without significant changes in the apparent dissociation constant (KD) of labeled hormone for its specific receptor. In normal adult animals, thyroxine treatment, i.e. hyperthyroidism, had an opposite effect on the Bmax (25-50% increase), without changes in the KD. In developing PTU-treated rats, the abnormalities completely disappeared after therapy with increasing physiological doses of thyroxine; consequently they were directly related to thyroid deficiency and not to toxic effects of PTU. Moreover, the abnormalities resulting from induced hypothyroidism were reversible. In developing and adult hypothyroid rats, neither basal, NaF-, nor Gpp(NH)p-stimulated adenylate cyclase activities were significantly affected. Glucagon-sensitive adenylate cyclase activity seemed to be slightly increased (by about 15%), without changes in the apparent activation constant (Kact). These results are considered in parallel with findings on plasmatic glucagon and vasopressin levels, compared with similar previous reports related to renal vasopressin receptors, and discussed with respect to unpublished observations concerning hepatic responsiveness to glycogenolytic hormones in young and adult rats with induced hypothyroidism.

Reduction in hepatic but not in renal and vascular vasopressin receptor number in hyperinsulinemic mice and rats.

Hepatic plasma membranes of female obese mice C57 BL-6 orl ob/ob (ob/ob mice) completely lack vasopressin (VP) receptors of the V1 type whereas kidney VP receptors are normally expressed and functionally coupled to adenylate cyclase. To discover if these alterations are linked to a genetic defect of the V1 receptor, we have studied the binding of VP on liver and kidney membranes of two other models, female diabetic mice C57 BL-6 orl db/db (db/db mice) and female Zucker rats Fatty/orl fa/fa (fa/fa rats), which exhibit different temporal pattern of obesity, hyperinsulinemia and insulin resistance. In addition, since VP is known to exert its vascular response through stimulation of V1 receptors, we have studied the reactivity of VP of isolated tail artery in the three different models, ob/ob and db/db mice and fa/fa rats, and in their respective controls. In all cases, VP kidney receptors and VP vascular reactivity are normal. db/db mice exhibit a marked decrease in hepatic VP receptors whereas a 50% decrease was observed in 32 week fa/fa rats. Angiotensin II and prazosin binding sites are still present as well as the adenylate cyclase response to glucagon. These results suggest that the specific alteration in liver VP receptors is not related to a defect in V1 receptor genetic expression but is specific for liver and appears to parallel the level of hyperinsulinemia and/or insulin resistance.

Human IgE response to the administration of blood components. II. Repeated gammaglobulin injections.

43 adults from a renal dialysis unit staff have received regularly spaced gamma-globulin administrations for hepatitis B prophylaxis. Several blood samples were collected over a prolonged period of time (160 days). Following gamma-globulin administration, anti-immunoglobulin antibodies of the IgE class were detected in 80% of this population, a fortnight after the first injection using serum absorptions on polymerized gamma-globulins or a specific inverse RAST method. The reactivity pattern of these IgE anti-immunoglobulin antibodies was similar to that observed for the anti-immunoglobulin antibodies with "limited specificity" detected by passive hemagglutination, in that they reacted with only one of the immunoglobulins of the panel used for their detection. A decrease of the overall IgE levels was observed in 62% of the subjects for a prolonged period of time following gamma-globulin administration. This suggests a feedback regulation mechanism for the reagin production in man, as it has already been observed in animals. A high incidence of anti-immunoglobulin antibodies of various classes was observed in this study. However, only a small number (4/43) of adverse reactions appeared following gamma-globulin administration. For some of these subjects, the presence of specific IgE anti-immunoglobulin, detected by the inverse-RAST technique, suggests a possible role of such antibodies in some intolerance reactions to gamma-globulin administration.

[Application of a reversed passive hemagglutination test to the detection of HBs antigen]. / Technique d'hémagglutination passive inverse appliquée à la détection de l'antigène HBs

In this report we present an evaluation of the sensitivity, specificity and ability to detect HBs Ag carriers of a new reversed passive hemagglutination test, using immunochemically purified chimpanzee anti HBs bound to stabilized human erythrocytes. The method was shown to have a sensitivity equal (within one two fold dilution) to that of the Ausria I 125 ratio immuno assay, and in a double blind comparison detected essentially the same number of Hbs Ag containing specimens among volunteer blood donors. The method therefore provides an economical method for the third generation testing of blood donors. The methodology which has been described incorporates a definitive specificity test in which serum drawn before and after immunization of chimpanzees with purified HBs Ag is compared for its ability to neutralize the hemagglutination reaction. The use of serum from the same animal for this purpose avoids the theoretical possibility that antiglobulin antibodies directed at subclass determinants such as Gm of Inv could be differentially inhibited due to possible subclass differences in the blocking sera employed. A reliable test for specificity of HBs Ag screening results is essential to avoid false notification of donors that they are carriers of hepatitis B virus.

A new reversed passive hemagglutination test for detection of HBsAg.

A new reversed passive hemagglutination test for HBsAg, termed Raphadex B, has been developed using immunochemically purified chimpanzee anti-HBs bound to stabilized human erythrocytes. The test has been found to have equivalent sensitivity to the Ausria 125I radioimmunoassay, and detected a similar number of HBsAg-containing specimens in screening of volunteer blood donors. This method offers an economical approach to third generation methodology for hepatitis B screening of blood donors.