Bone Is Not Essential for Osteoclast Activation

Background

The mechanism whereby bone activates resorptive behavior in osteoclasts, the cells that resorb bone, is unknown. It is known that α_vβ₃ ligands are important, because blockade of α_vβ₃ receptor signaling inhibits bone resorption, but this might be through inhibition of adhesion or migration rather than resorption itself. Nor is it known whether α_vβ₃ ligands are sufficient for resorption the consensus is that bone mineral is essential for the recognition of bone as the substrate appropriate for resorption.

Methodology/Principal Findings

Vitronectin- but not fibronectin-coated coverslips induced murine osteoclasts to secrete tartrate-resistant acid phosphatase, as they do on bone. Osteoclasts incubated on vitronectin, unlike fibronectin, formed podosome belts on glass coverslips, and these were modulated by resorption-regulating cytokines. Podosome belts formed on vitronectin-coated surfaces whether the substrates were rough or smooth, rigid or flexible. We developed a novel approach whereby the substrate-apposed surface of cells can be visualized in the scanning electron microscope. With this approach, supported by transmission electron microscopy, we found that osteoclasts on vitronectin-coated surfaces show ruffled borders and clear zones characteristic of resorbing osteoclasts. Ruffles were obscured by a film if cells were incubated in the cathepsin inhibitor E64, suggesting that removal of the film represents substrate-degrading behavior. Analogously, osteoclasts formed resorption-like trails on vitronectin-coated substrates. Like bone resorption, these trails were dependent upon resorbogenic cytokines and were inhibited by E64. Bone mineral induced actin rings and surface excavation only if first coated with vitronectin. Fibronectin could not substitute in any of these activities, despite enabling adhesion and cell spreading.

Conclusions/Significance

Our results show that ligands α_vβ₃ are not only necessary but sufficient for the induction of resorptive behavior in osteoclasts; and suggest that bone is recognized through its affinity for these ligands, rather than by its mechanical or topographical attributes, or through a putative ‘mineral receptor’.     

Benzodiazepine binding to mitochondrial membranes of the amoeba Acanthamoeba castellanii and the yeast Saccharomyces cerevisiae

Benzodiazepine binding sites were studied in mitochondria of unicellular eukaryotes, the amoeba Acathamoeba castellanii and the yeast Saccharomyces cerevisiae, and also in rat liver mitochondria as a control. For that purpose we applied Ro5-4864, a well-known ligand of the mitochondrial benzodiazepine receptor (MBR) present in mammalian mitochondria. The levels of specific [(3)H]Ro5-4864 binding, the dissociation constant (K(D)) and the number of [(3)H]Ro5-4864 binding sites (B(max)) determined for fractions of the studied mitochondria indicate the presence of specific [(3)H]Ro5-4864 binding sites in the outer membrane of yeast and amoeba mitochondria as well as in yeast mitoplasts. Thus, A. castellanii and S. cerevisiae mitochondria, like rat liver mitochondria, contain proteins able to bind specifically [(3)H]Ro5-4864. Labeling of amoeba, yeast and rat liver mitochondria with [(3)H]Ro5-4864 revealed proteins identified as the voltage dependent anion selective channel (VDAC) in the outer membrane and adenine nucleotide translocase (ANT) in the inner membrane. Therefore, the specific MBR ligand binding is not confined only to mammalian mitochondria and is more widespread within the eukaryotic world. However, it can not be excluded that MBR ligand binding sites are exploited efficiently only by higher multicellular eukaryotes. Nevertheless, the MBR ligand binding sites in mitochondria of lower eukaryotes can be applied as useful models in studies on mammalian MBR.     

The prion peptide forms ion channels in planar lipid bilayers

One of the hypotheses concerning the pathogenic properties of the prion protein considers its influence on cellular ion homeostasis. Using the lipid bilayer technique, the influence of prion-derived peptides on the lipid bilayer conductance was characterized. To evaluate the physiological significance and possible pathological functions of the peptides, their effect on the membrane potential and respiration rate of hippocampal mitochondria was also studied. We used a peptide bearing the human prion protein sequence YSNQNNF (PrP [169-175]), and peptide SSQNNF (PrP [170-175]) bearing a naturally-occurring mutation in position 171 [N(r)S] linked to schizoaffective diseases in humans (Samaia, H.B., Mari, J.J., Vallada, H.P., Moura R.P., Simpson A.J.G., Brentani R.R. A prion-linked psychiatric disorder. Nature 390 (1997) 241). In this report, we show that PrP [170-175] N171S increases the conductance of planar lipid bilayers. Based on the conductance of single channel currents recorded in 500/500 mM KCl (cis/trans), we found a single channel conductance of 8 to 26 pS. The native prion peptide PrP [169-175] does not form ion channels in the lipid bilayer. Neither of the peptides significantly changed the membrane potential or respiration rate of isolated rat hippocampal mitochondria. We propose a possible mechanism for channel formation by aggregation of the prion-derived peptide.     

The expansion of CD4<Superscript>+</Superscript>CD28<Superscript>-</Superscript> T cells in patients with rheumatoid arthritis

Clonal expansion of CD4⁺CD28^- T cells is a characteristic finding in patients with rheumatoid arthritis (RA). Expanded CD4⁺ clonotypes are present in the peripheral blood, infiltrate into the joints, and persist for years. CD4⁺CD28^- T cells are oligoclonal lymphocytes that are rare in healthy individuals but are found in high percentages in patients with chronic inflammatory diseases. The size of the peripheral blood CD4⁺CD28^- T-cell compartment was determined in 42 patients with RA and 24 healthy subjects by two-color FACS analysis. The frequency of CD4⁺CD28^- T cells was significantly higher in RA patients than in healthy subjects. Additionally, the number of these cells was significantly higher in patients with extra-articular manifestations and advanced joint destruction than in patients with limited joint manifestations. The results suggest that the frequency of CD4⁺CD28^- T cells may be a marker correlating with extra-articular manifestations and joint involvement.     

Crystal structure of a dual activity IMPase/FBPase (AF2372) from Archaeoglobus fulgidus. The story of a mobile loop

Several hyperthermophilic organisms contain an unusual phosphatase that has dual activity toward inositol monophosphates and fructose 1,6-bisphosphate. The structure of the second member of this family, an FBPase/IMPase from Archaeoglobus fulgidus (AF2372), has been solved. This enzyme shares many kinetic and structural similarities with that of a previously solved enzyme from Methanococcus jannaschii (MJ0109). It also shows some kinetic differences in divalent metal ion binding as well as structural variations at the dimer interface that correlate with decreased thermal stability. The availability of different crystal forms allowed us to investigate the effect of the presence of ligands on the conformation of a mobile catalytic loop independently of the crystal packing. This conformational variability in AF2372 is compared with that observed in other members of this structural family that are sensitive or insensitive to submillimolar concentrations of Li(+). This analysis provides support for the previously proposed mechanism of catalysis involving three metal ions. A direct correlation of the loop conformation with strength of Li(+) inhibition provides a useful system of classification for this extended family of enzymes.     

Evaluation of selected features of Staphylococcus cohnii enabling colonization of humans

Based on iron utilization, sensitivity to skin fatty acids, lipolytic and proteolytic activity the potential abilities ofStaphylococcus cohnii strains to colonize humans were evaluated. The investigation included 60 strains that belong to both subspecies,viz. S. cohnii ssp.cohnii andS. cohnii ssp.urealyticus. Strains were isolated from different sources of theIntensive Care Unit and from non-hospital environment. Most of the strains were multiple antibiotic-resistant. Strains of both subspecies revealed a relatively low iron requirement. These strains were capable of utilizing iron bound in oxo acids and from host iron-binding proteins.S. cohnii ssp.urealyticus were more effective in iron uptake thanS. cohnii ssp.cohnii. All investigated strains revealed sensitivity to skin fatty acids, butS. cohnii ssp.urealyticus strains were more resistant. Special features of strains of this subspecies promote colonization of humans.     

Structural basis for CO2 fixation by a novel member of the disulfide oxidoreductase family of enzymes, 2-ketopropyl-coenzyme M oxidoreductase/carboxylase

The NADPH:2-ketopropyl-coenzyme M oxidoreductase/carboxylase (2-KPCC) is the terminal enzyme in a metabolic pathway that results in the conversion of propylene to the central metabolite acetoacetate in Xanthobacter autotrophicus Py2. This enzyme is an FAD-containing enzyme that is a member of the NADPH:disulfide oxidoreductase (DSOR) family of enzymes that include glutathione reductase, dihydrolipoamide dehydrogenase, trypanothione reductase, thioredoxin reductase, and mercuric reductase. In contrast to the prototypical reactions catalyzed by members of the DSOR family, the NADPH:2-ketopropyl-coenzyme M oxidoreductase/carboxylase catalyzes the reductive cleavage of the thioether linkage of 2-ketopropyl-coenzyme M, and the subsequent carboxylation of the ketopropyl cleavage product, yielding the products acetoacetate and free coenzyme M. The structure of 2-KPCC reveals a unique active site in comparison to those of other members of the DSOR family of enzymes and demonstrates how the enzyme architecture has been adapted for the more sophisticated biochemical reaction. In addition, comparison of the structures in the native state and in the presence of bound substrate indicates the binding of the substrate 2-ketopropyl-coenzyme M induces a conformational change resulting in the collapse of the substrate access channel. The encapsulation of the substrate in this manner is reminiscent of the conformational changes observed in the well-characterized CO2-fixing enzyme ribulose 1,5-bisphosphate carboxylase/oxidase (Rubisco).     

The metabolic heterogeneity of pathogenic and nonpathogenic coagulase negative staphylococci

The coagulase negative staphylococci are a metabolically heterogenous group of strains. The investigated pathogenic and nonpathogenic strains differed markedly in respiration activity and character of endogenous metabolism.     

Crystal structure of alpha-hordothionin at 1.9 Angstrom resolution

Crystal structure of ubiquitous toxin from barley alpha-hordothionin (alpha-HT) has been determined at 1.9A resolution by X-ray crystallography. The primary sequence as well as the NMR solution structure of alpha-HT firmly established that alpha-HT belongs to a family of membrane active plant toxins-thionins. Since alpha-HT crystallized in a space group (P4(1)2(1)2) that is different from the space group (I422) of previously determined alpha(1)- and beta-purothionins, and visocotoxin A3, therefore, it provided independent information on protein-protein interactions that may be relevant to the toxin mechanism. The structure of alpha-HT not only confirms overall architectural features (crambin fold) but also provides an additional confirmation of the role for crucial solute molecules, that were postulated to be directly involved in the mechanism of toxicity for thionins.     

Mast cells in the mouse mammary gland--correlation with the development of lactiferous structures

Specimens of mouse mammary glands obtained from animals being in different phases of reproductive cycle were collected. After staining MCN, the total mast cell area (TMC), mean mast cell area (MMC), and lactiferous structure density index (LDI) were examined in sections, using a computer image analysis system. Statistically significant (P < 0.05) results were: 1. An increase in MCN observed in Group I (4-5 and 9-10 days of pregnancy), a decrease in MCN observed in Group II (2nd and 10th day of lactation); 2. Changes of TMC fully corresponding to changes of MCN; 3. Increase in MMC observed in Group I at mid (days 9-10) and at the end (days 18-19) of pregnancy, decrease in MMC observed in Group IIB (10th day of lactation); 4. LDI (%) higher at the end of pregnancy (Group IC) and during lactation (groups IIA, IIB), compared with control (23.5 +/- 4.12, 37.6 +/- 3.24, 71.0 +/- 4.33 vs. 3.8 +/- 0.39). The observed changes in the number and size of MC strictly correspond to physiological phenomena leading to alternation of the mouse mammary gland functional status by development/involution of the lactiferous structures.