The influence of 1-hydroxyethane-1,1-diphosphonate and dichloromethanediphosphonate on lysine hydroxylation and cross-link formation in rat bone, cartilage and skin collagen.

The effects in vivo of dichloromethanediphosphonate and 1-hydroxyethane 1,1-diphosphonate on collagen solubility, hydroxylation of lysine and proline and on the formation of collagen intermolecular cross-links were studied by using rat bone, cartilage and skin tissues. Dichloromethanediphosphonate decreased bone collagen solubility both in acetic acid and after pepsin treatment. Although none of the diphosphonates had any effect on the hydroxylation of proline, dichloromethane-diphosphonate, but not 1-hydroxyethane-1,1-diphosphonate, increased the number of hydroxylysine residues in the alpha-chains of bone, skin and cartilage collagen. The stimulatory effect was dose-dependent. The dichloromethanediphosphonate-mediated increase in hydroxylysine residues in bone and cartilage was manifested in an increase of dihydroxylysinonorleucine, the cross-link that is formed by the condensation of two hydroxylysine residues. The cross-link hydroxylysinonorleucine, a condensation product of hydroxylysine and lysine, on the other hand, was decreased. The total number of intermolecular cross-links was not changed by the diphosphonate.     

The uptake and metabolism of [32P]pyrophosphate by mouse calvaria in vitro

In order to study the uptake and metabolism of PP(i) by bone, (32)PP(i) was added to the medium surrounding explanted mouse calvaria maintained in organ culture. Most of the PP(i) was hydrolysed during incubation, but there was a measurable entry of intact PP(i) into bone. When (32)P(i) was added to the medium, synthesis of PP(i) and organic phosphates from P(i) was observed in bone. There was no detectable passage of PP(i) from bone into the medium. These results are discussed in terms of two models of pyrophosphate hydrolysis and exchange. Some quantitative estimates about the fate of PP(i) in bone were made.     

Aggregation of hydroxyapatite crystals

A system to study the aggregation of hydroxyapatite crystals was developed. The effect of several factors (Ca²⁺ × P_i product, Ca²⁺ /P_i ratio, pH, and various substances) were tested. Pb²⁺, Zn²⁺, Mg²⁺ and methyleneblue had only small effects; citrate inhibited aggregation. Pyrophosphate was a strong inhibitor and the diphosphonates disodium ethane-1-hydroxy-1,1-diphosphonate and disodium duchloromethylene diphosphonate were even more potent. The monophosphonate pentanemonophosphonate had no effect. Potent inhibition also occurred with glycosaminoglycans: heparin > hyaluronic acid > dermatan sulfate > chondroitin 4-sulfate > chondroitin 6-sulfate. Urine also showed high inhibitory activity. The inhibition of heparin but not that of hyaluronic acid, PP_i or urine was abolished by egg white lysozyme. The effects described might be relevant in the normal mineralization process as well as in the mechanisms leading to pathological calcification, such as urinary stone formation.     

Calcification inhibitors in rat and human serum and plasma

Rat and human serum and plasma were shown to contain considerable amounts of calcium phosphate precipitation inhibitors. Two general classes of inhibiting molecules were observed for both species: high molecular weight (approx. 30 000-200 000) and low molecular weight (less than 1000). The high molecular weight components eluted from a Bio-Gel P-200 column in two peaks, one at approx. 158 000 and a broader peak at approx. 43 000. The identity of these inhibitors is unknown at present. Low molecular weight inhibitors include magnesium, pyrophosphate, and citrate ions and at least one unidentified component that coelutes with pyrophosphate and citrate on a Bio-Gel P-4 column. Quantitatively, most of the inhibitor activity resides in the high molecular weight components and it is possible that it is this activity which is responsible for maintaining the metastability of the circulating fluids. The role of the low molecular weight components may be to regulate calcification at sites inaccessible to high molecular weight molecules.     

Role of matrix vesicles in calcification.

The role of matrix vesicles in the calcification process was investigated in vitro. Isolated vesicles were unable to transport calcium actively. The ATPase activity was not stimulated by calcium in the presence of an optimal magnesium concentration. At a physiological substrate concentration of pyrophosphate, the pyrophosphatase had a pH optimum around 7.0. The vesicles nucleated calcium phosphate precipitation independently of the presence of hydrolyzable phosphate compounds. It is suggested that vesicles induce calcification by nucleating calcium phosphate precipitation and through the local destruction of pyrophosphate, a crystallization inhibitor.     

Spatial regulation of Aurora A activity during mitotic spindle assembly requires RHAMM to correctly localize TPX2

Construction of a mitotic spindle requires biochemical pathways to assemble spindle microtubules and structural proteins to organize these microtubules into a bipolar array. Through a complex with dynein, the receptor for hyaluronan-mediated motility (RHAMM) cross-links mitotic microtubules to provide structural support, maintain spindle integrity, and correctly orient the mitotic spindle. Here, we locate RHAMM to sites of microtubule assembly at centrosomes and non-centrosome sites near kinetochores and demonstrate that RHAMM is required for the activation of Aurora kinase A. Silencing of RHAMM delays the kinetics of spindle assembly, mislocalizes targeting protein for XKlp2 (TPX2), and attenuates the localized activation of Aurora kinase A with a consequent reduction in mitotic spindle length. The RHAMM–TPX2 complex requires a C-terminal basic leucine zipper in RHAMM and a domain that includes the nuclear localization signal in TPX2. Together, our findings identify RHAMM as a critical regulator for Aurora kinase A signaling and suggest that RHAMM ensures bipolar spindle assembly and mitotic progression through the integration of biochemical and structural pathways.     

No inbreeding depression in laboratory‐reared individuals of the parasitoid wasp Allotropa burrelli

Inbreeding depression is a major concern in almost all human activities relating to plant and animal breeding. The biological control of pests with natural enemies is no exception, because populations of biocontrol agents experience a series of bottlenecks during importation, rearing, and introduction. A classical biological control program for the Comstock mealybug Pseudococcus comstocki (Hemiptera: Pseudococcidae) was initiated in France in 2008, based on the introduction of an exotic parasitoid, Allotropa burrelli Mues. (Hymenoptera: Platygastridae), a haplodiploid parasitoid imported from Japan. We evaluated the sensitivity of A. burrelli to inbreeding, to optimize rearing and release strategies. We compared several morphological and life‐history traits between the offspring of siblings and the offspring of unrelated parents. We took into account the low level of genetic variability due to the relatively small size of laboratory‐reared populations by contrasting two types of pedigree: one for individuals from a strain founded from a single field population, and the other generated by hybridizing individuals from two strains founded from two highly differentiated populations. Despite this careful design, we obtained no evidence for a negative impact of inbreeding on laboratory‐reared A. burrelli. We discussed the results in light of haplodiploid sex determination and parasitoid mating systems, and classical biological control practices.