Studies of the mode of action of the bisphosphonate alendronate showed that 1 d after the injection of 0.4 mg/kg [3Hjalen-dronate to newborn rats, 72% of the osteoclastic surface, 2% of the bone forming, and 13% of all other surfaces were densely labeled. Silver grains were seen above the osteoclasts and no other cells. 6 d later the label was 600-1,000 itm away from the epiphyseal plate and buried inside the bone, indicating normal growth and matrix deposition on top of alendronate-containing bone. Osteoclasts from adult animals, infused with parathyroid hormone-related peptide (1-34) and treated with 0.4 mg/kg alendronate subcutaneously for 2 d, all lacked ruffled border but not clear zone.In vitro alendronate bound to bone particles with a Kd of -1 mM and a capacity of 100 nmol/mg at pH 7. At pH 3.5 binding was reduced by 50%. Alendronate inhibited bone resorption by isolated chicken or rat osteoclasts when the amount on the bone surface was around 1.3 X 10-3 fmol/Mm2, which would produce a concentration of 0.1-1 mM in the resorption space if 50% were released. At these concentrations membrane leakiness to calcium was observed. These findings suggest that alendronate binds to resorption surfaces, is locally released during acidification, the rise in concentration stops resorption and membrane ruffling, without destroying the osteoclasts. (J.

No abstract

SummaryMatrix vesicles have been implicated in the mineralization of calcified cartilage, bone and dentin for more than 40 years. During this period, their exact role, if any in the nucleation of hydroxyapatite mineral, and its subsequent association with the collagen fibrils in the organic matrix has been debated and remains controversial. Several hypotheses have been recently introduced to explain in greater detail how matrix vesicles function in biomineralization. This review will summarize recent advances, and open questions in the ongoing saga of these interesting and enigmatic extracellular organelles.

Annexins II, V, and VI are major components of matrix vesicles (MV), i.e. particles that have the critical role of initiating the mineralization process in skeletal tissues. Furthermore, types II and X collagen are associated with MV, and these interactions mediated by annexin V stimulate Ca 2؉ uptake and mineralization of MV. However, the exact roles of annexin II, V, and VI and the interaction between annexin V and types II and X collagen in MV function and initiation of mineralization are not well understood. Annexins are a family of proteins that has in common the ability to bind to acidic phospholipids in the presence of Ca 2ϩ (1, 2). The family consists of at least 12 members, and three of them, annexins II, V, and VI, are highly expressed in calcifying cartilage and bone (3, 4). Annexin II and V each contain four 70 -80 amino acids repeats with an annexin consensus sequence. Annexin VI contains eight such repeats. These four or eight repeats form the conserved core region, which is responsible for the Ca 2ϩ -dependent binding of the proteins to phospholipids. In contrast, the N-terminal regions of the annexins are highly variable and may contribute to the specific functions of the various annexins (1, 2).Annexins II, V, and VI are major components of matrix vesicles (MV), 1 which are particles that, after being released from the plasma membrane of hypertrophic chondrocytes or osteoblasts, have the critical role of initiating the mineralization process in cartilage and perhaps in bone (3, 5). Three independent lines of evidence indicate that annexin II, V, and VI exhibit distinct Ca 2ϩ ion channel properties. First, when inserted into artificial phosphatidylserine bilayers they form voltage-dependent Ca 2ϩ ion channels (6 -8). Second, the crystal structures of these annexins are largely ␣-helical with parallel barrels of ␣-helical domains forming a hydrophilic, charged pore through the center of the protein (6, 8, 9). Third, annexin II, V, and VI are able to mediate Ca 2ϩ influx into artificial liposomes (10;11). It was shown that annexin-mediated Ca 2ϩ influx into liposomes is rapid during the first 20 min and then reaches a plateau after 20 min (10, 11).The initial phase of MV-mediated mineralization is characterized by the uptake of mineral ions by these particles and the formation and growth of the first mineral phase inside the vesicles (5). Because MV are enclosed by a membrane, channel proteins are required to mediate the influx of mineral ions into these particles. Previous findings from our and other laboratories, showed that chymotrypsin treatment, which removes most of the annexins from MV, and zinc treatment, which inhibits annexin-mediated Ca 2ϩ influx into phosphatidylserine (PS)-enriched liposomes, diminished MV Ca 2ϩ uptake (12-15), suggesting that annexins II, V, and VI serve as ion channels in MV, enabling Ca 2ϩ loading of the vesicles during the initial phase of mineralization.Previous studies have revealed that collagen types II and X are associated with the outer surface of MV (16). We...

Experiments were performed to determine whether beta-glycerophosphate (beta-GP) promoted mineralization in vitro by modulating bone cell metabolic activity and/or serving as a local source of inorganic phosphate ions (Pi). Using MC3T3-E1, ROS 17/2.8, and chick osteoblast-like cells in the presence of beta-GP or Pi, we examined mineral formation, lactate generation, alkaline phosphatase (AP) activity, and protein and phospholipid synthesis. Neither beta-GP nor Pi modulated any of the major biosynthetic activities of the bone cells. Thus, we found no change in the levels of phospholipids, and the total protein concentration remained constant. Measurement of lactate synthesis showed that beta-GP did not effect the rate of anaerobic glycolysis. Evaluation of medium Pi levels clearly indicated that beta-GP was hydrolyzed by bone cells; within 24 hours, almost 80% of 10 mM beta-GP was hydrolyzed. It is likely that this local increase in medium Pi concentration promoted rapid mineral deposition. Chemical, energy dispersive X-ray, and Fourier transform infrared analysis of the mineral formed in the presence of beta-GP showed that it was nonapatitic; moreover, mineral particles were also seen in the culture medium itself. Experiments performed with a cell-free system indicated that mineral particles formed spontaneously in the presence of AP and beta-GP and were deposited into a collagen matrix. We conclude that medium supplementation with beta-GP or Pi should not exceed 2 mM. If this value is exceeded, then there will be nonphysiological mineral deposition in the bone cell culture.

In normal healthy individuals, mineral formation is restricted to specialized tissues which form the skeleton and the dentition. Within these tissues, mineral formation is tightly controlled both in growth and development and in normal adult life. The mechanism of calcification in skeletal and dental tissues has been under investigation for a considerable period. One feature common to almost all of these normal mineralization mechanisms is the elaboration of matrix vesicles, small (20-200 nm) membrane particles, which bud off from the plasma membrane of mineralizing cells and are released into the pre-mineralized organic matrix. The first crystals which form on this organic matrix are seen in and around matrix vesicles. Pathologic ectopic mineralization is seen in a number of human genetic and acquired diseases, including calcification of joint cartilage resulting in osteoarthritis and mineralization of the cardiovasculature resulting in exacerbation of atherosclerosis and blockage of blood vessels. Surprisingly, increasing evidence supports the contention that the mechanisms of soft tissue calcification are similar to those seen in normal skeletal development. In particular, matrix vesicle-like membranes are observed in a number of ectopic calcifications. The purpose of this review is to describe how matrix vesicles function in normal mineral formation and review the evidence for their participation in pathologic calcification.

We previously defined eight groups of monoclonal antibodies which react with distinct epitopes of herpes simplex virus glycoprotein D (gD). One of these, group VII antibody, was shown to react with a type-common continuous epitope within residues 11 to 19 of the mature glycoprotein (residues 36 to 44 of the predicted sequence of gD). In the current investigation, we have localized the sites of binding of two additional antibody groups which recognize continuous epitopes of gD. The use of truncated forms of gD as well as computer predictions of secondary structure and hydrophilicity were instrumental in locating these epitopes and choosing synthetic peptides to mimic their reactivity. Group II antibodies, which are type common, react with an epitope within residues 268 to 287 of the mature glycoprotein (residues 293 to 312 of the predicted sequence). Group V antibodies, which are gD-l specific, react with an epitope within residues 340 to 356 of the mature protein (residues 365 to 381 of the predicted sequence). Four additional groups of monoclonal antibodies appear to react with discontinuous epitopes of gD-1, since the reactivity of these antibodies was lost when the glycoprotein was denatured by reduction and alkylation. Truncated forms of gD were used to localize these four epitopes to the first 260 amino acids of the mature protein. Competition experiments were used to assess the relative positions of binding of various pairs of monoclonal antibodies. In several cases, when one antibody was bound, there was no interference with the binding of an antibody from another group, indicating that the epitopes were distinct. However, in other cases, there was competition, indicating that these epitopes might share some common amino acids.

An antigenic determinant capable of inducing type-common herpes simplex virus (HSV)-neutralizing antibodies has been located on glycoprotein D (gD) of HSV type 1 (HSV-1). A peptide of 16 amino acids corresponding to residues 8 to 23 of the mature glycoprotein (residues 33 to 48 of the predicted gD-1 sequence) was synthesized. This peptide reacted with an anti-gD monoclonal antibody (group VII) previously shown to neutralize the infectivity of HSV-1 and HSV-2. The peptide was also recognized by polyclonal antibodies prepared against purified gD-1 but was less reactive with anti-gD-2 sera. Sera from animals immunized with the synthetic peptide reacted with native gD and neutralized both HSV-1 and HSV-2. Herpes simplex viruses (HSVs) cause a number of human diseases, including cold sores, eye and genital infections, and encephalitis (21). HSV glycoproteins are structural components of the virion envelope and have been implicated in virus-induced alterations of mammalian cells (22, 27). These glycoproteins are expressed on infected cell plasma membranes and act as major antigenic stimuli for the cellular and humoral responses of the host (22, 27). Four virion glycoproteins, designated gB, gC, gD, and gE have been described (26). One of these, glycoprotein D (gD), apparently plays a major role in the immune response to HSV. Evidence to support this suggestion is as follows. (i) Purified gD of HSV type 1 (oral; HSV-1) (gD-1) or HSV-2 (genital) (gD-2) stimulates high titers of type-common virus-neutralizing antibody (6, 30); (ii) passive immunization with monoclonal antibodies directed against gD protects mice from challenge by a lethal dose of HSV (3, 11, 18); (iii) gD mediates antibody-dependent, complement-mediated cytotoxicity and antibody-dependent, cell-mediated cytotoxicity (3, 23, 25); (iv) purified gD is able to protect mice against lethal challenge with either HSV-1 or HSV-2 (D. Long, T.