Mice over-expressing salmon calcitonin have strongly attenuated osteoarthritic histopathological changes after destabilization of the medial meniscus.

OBJECTIVE: Calcitonin is well-known for its inhibitory actions on bone-resorbing osteoclasts and recently potential beneficial effects on cartilage were shown. We investigated effects of salmon calcitonin (sCT) on the articular cartilage and bone, after destabilization of the medial meniscus (DMM) in normal and sCT over-expressing mice. DESIGN: Bone phenotype of transgenic (TG) C57Bl/6 mice over-expressing sCT at 6 months and 12 months was investigated by (1) serum osteocalcin and urinary deoxypyridinoline and (2) dynamic and normal histomorphometry of vertebrae bodies. In subsequent evaluation of cartilage and subchondral bone changes, 44 10-week old TG or wild-type (WT) mice were randomized into four groups and subjected to DMM or sham-operations. After 7 weeks animals were sacrificed, and knee joints were isolated for histological analysis. RESULTS: Trabecular bone volume (BV/TV) increased 150% after 6 months and 300% after 12 months in sCT-expressing mice when compared to WT controls (P<0.05). Osteoblast number, bone formation rate and osteocalcin measurements were not affected in TG mice over-expressing sCT. In WT animals, a 5-fold increase in the quantitative erosion index was observed after DMM, and the semi-quantitative OARSI score showed over 400% (P<0.001) increase, compared to sham-operated WT mice. DMM-operated TG mice were protected against cartilage erosion and showed a 65% and 64% (P<0.001) reduction, respectively, for the two histopathological evaluation methods. CONCLUSIONS: sCT over-expressing mice had higher bone volume, and were protected against cartilage erosion. These data suggest that increased levels of sCT may hamper the pathogenesis of osteoarthritis (OA). However more studies are necessary to confirm these preliminary results.

Malondialdehyde-altered protein occurs in atheroma of Watanabe heritable hyperlipidemic rabbits.

It has been proposed that chemically reactive lipids released during lipid peroxidation convert low density lipoprotein (LDL), the major carrier of plasma cholesterol, to an abnormal form and that receptor-mediated clearance of this altered LDL produces cholesteryl ester deposition in macrophage-derived foam cells of atheroma. Immuno-cytochemical analyses now reveal the presence of protein modified by malondialdehyde, a peroxidative end product, which colocalizes with the extracellular deposition of apolipoprotein B-100 protein of LDL in atheroma from Watanabe heritable hyperlipidemic rabbits. These findings provide direct evidence for the existence in vivo of protein modified by a physiological product of lipid peroxidation within arterial lesions.

Receptor recognition of maleyl-albumin induces chemotaxis in human monocytes.

We demonstrate here that the exceptionally active maleyl-albumin receptor of human monocytes functions in vitro as a chemoattractant receptor. Chemotaxis of human monocytes occurs at an effective median dose of 3-4 microM maleyl-albumin, a concentration representing 1% of the total albumin in the adult human. Computerized analyses by LIGAND of the saturable binding of maleyl-albumin to human monocytes reveal two classes of binding sites, described by dissociation constants of 37 nM and 5.3 microM with maximal binding of 1.6 and 23 pmol maleyl-albumin/mg cellular protein, respectively. Chemotaxis of human monocytes thus occurs at concentrations of maleyl-albumin promoting binding to the lower-affinity sites. We propose that conformational isomers of albumin that are chemotactic may form in vivo and that albumin, in addition to receptor-independent plasma transport functions, may also play an important role in the receptor-mediated recruitment and accumulation of phagocytic cells at sites of inflammation and injury.

Two distinct receptors account for recognition of maleyl-albumin in human monocytes during differentiation in vitro.

A comparison of the receptor-mediated interaction of malondialdehyde-low density lipoprotein and maleyl-albumin has been examined in human monocytes during differentiation in vitro. The recognition of both ligands by the scavenger receptor of these cells has been confirmed. We now report that human monocytes express a second cellular surface receptor for maleyl-albumin that is distinct from the scavenger receptor. The activity of the maleyl-albumin receptor, determined by both binding and lysosomal hydrolytic assays, substantially exceeds that of the scavenger receptor in freshly isolated monocytes. A dramatic and rapid decline in the activity of the maleyl-albumin receptor occurs within 72 to 96 h during differentiation in vitro. At day 7, while only 5-10% of the original activity of the maleyl-albumin receptor remains, it is similar to that of the maximally expressed scavenger receptor. Both the binding and hydrolysis of ligand mediated by the maleyl-albumin receptor are specifically inhibited by alpha-casein and alkaline-treated albumin; neither of these proteins is recognized by the scavenger receptor. The occurrence of the exceptionally active maleyl-albumin receptor on freshly isolated human monocytes suggests that it participates in processes necessary to the function of the cells that diminish in importance after differentiation of the monocytes into macrophages in vitro. Furthermore, while maleyl-albumin is a useful adjunct to studies of cellular events mediated by the scavenger receptor, the presence of a second receptor for maleyl-albumin must be taken into account as a potential contributing and complicating event.

Rabbit beta-migrating very low density lipoprotein increases endothelial macromolecular transport without altering electrical resistance.

Rabbit aortic endothelial cells (RAEC) were grown on micropore filters in a new device. This system allowed in situ measurement of transendothelial electrical resistance (TEER). The monolayers demonstrated a TEER of 14 +/- 1 omega X cm2 at confluence. No difference was seen in the transport of low density lipoproteins (LDL) across endothelial cell monolayers obtained from normal or Watanabe heritable hyperlipidemic rabbits, indicating that the LDL receptor was not involved in the LDL transport. TEER was inversely correlated with 22Na transport (r2 = 0.93, P = less than 0.001) but not with 125I-LDL transport. The amount of LDL transported at 15 degrees C or across glutaraldehyde-fixed monolayers was half that of the controls at 37 degrees C. Preincubation of the monolayers with rabbit beta-migrating very low density lipoproteins (beta-VLDL) increased cholesterol content by 65%, and the transport of albumin and LDL doubled without a change in TEER. Removal of beta-VLDL from the culture medium resulted in the return of cellular cholesterol content and LDL transport to control values. We conclude that preincubation of RAEC with beta-VLDL resulted in an increased permeability to LDL and albumin, and that beta-VLDL may promote increased transendothelial transport of macromolecules in cholesterol-fed rabbits.

Endothelial responses to oxidized lipoproteins determine genetic susceptibility to atherosclerosis in mice.

BACKGROUND: Oxidized LDL has been found within the subendothelial space, and it exhibits numerous atherogenic properties, including induction of inflammatory genes. We examined the possibility that variations in endothelial response to minimally modified LDL (MM-LDL) constitute one of the genetic components in atherosclerosis. METHODS AND RESULTS: By a novel explant technique, endothelial cells (ECs) were isolated from the aorta of inbred mouse strains with different susceptibilities to diet-induced atherosclerosis. Responses to MM-LDL were evaluated by examining the expression of inflammatory genes involved in atherosclerosis, including monocyte chemotactic protein-1 (MCP-1) and macrophage-colony-stimulating factor (M-CSF), an oxidative stress gene, heme oxygenase-1 (HO-1), and other, noninflammatory, genes. ECs from the susceptible mouse strain C57BL/6J exhibited dramatic induction of MCP-1, M-CSF, and HO-1, whereas ECs from the resistant strain C3H/HeJ showed little or no induction. In contrast, ECs from the 2 strains responded similarly to lipopolysaccharide. CONCLUSIONS: These data provide strong evidence that genetic factors in atherosclerosis act at the level of the vessel wall.

On extracting design principles from biology: I. Method-General answers to high-level design questions for bioinspired robots.

When millions of years of evolution suggest a particular design solution, we may be tempted to abandon traditional design methods and copy the biological example. However, biological solutions do not often translate directly into the engineering domain, and even when they do, copying eliminates the opportunity to improve. A better approach is to extract design principles relevant to the task of interest, incorporate them in engineering designs, and vet these candidates against others. This paper presents the first general framework for determining whether biologically inspired relationships between design input variables and output objectives and constraints are applicable to a variety of engineering systems. Using optimization and statistics to generalize the results beyond a particular system, the framework overcomes shortcomings observed of ad hoc methods, particularly those used in the challenging study of legged locomotion. The utility of the framework is demonstrated in a case study of the relative running efficiency of rotary-kneed and telescoping-legged robots.

On extracting design principles from biology: II. Case study-the effect of knee direction on bipedal robot running efficiency.

Comparing the leg of an ostrich to that of a human suggests an important question to legged robot designers: should a robot's leg joint bend in the direction of running ('forwards') or opposite ('backwards')? Biological studies cannot answer this question for engineers due to significant differences between the biological and engineering domains. Instead, we investigated the inherent effect of joint bending direction on bipedal robot running efficiency by comparing energetically optimal gaits of a wide variety of robot designs sampled at random from a design space. We found that the great majority of robot designs have several locally optimal gaits with the knee bending backwards that are more efficient than the most efficient gait with the knee bending forwards. The most efficient backwards gaits do not exhibit lower touchdown losses than the most efficient forward gaits; rather, the improved efficiency of backwards gaits stems from lower torque and reduced motion at the hip. The reduced hip use of backwards gaits is enabled by the ability of the backwards knee, acting alone, to (1) propel the robot upwards and forwards simultaneously and (2) lift and protract the foot simultaneously. In the absence of other information, designers interested in building efficient bipedal robots with two-segment legs driven by electric motors should design the knee to bend backwards rather than forwards. Compared to common practices for choosing robot knee direction, application of this principle would have a strong tendency to improve robot efficiency and save design resources.

Lipoprotein (a) displays increased accumulation compared with low-density lipoprotein in the murine arterial wall.

Lipoprotein (a) (Lp(a)) is known to be an independent risk factor for cardiovascular disease, but the mechanisms by which it contributes to this disease remain unclear. Current evidence indicates that the closely related plasma particle, low-density lipoprotein (LDL), may initiate atherosclerosis through deposition in the arterial wall. This study has compared the ability of both lipoproteins to enter and accumulate within the arterial wall. Experiments were conducted in vivo with animals from two strains of mice: C57BL/6 mice, which develop fatty streak lesions upon challenge by a high-fat diet, and C3H/HeJ mice, which are resistant to lesion formation. Animals from both strains were maintained up to 16 weeks either on chow or high-fat diet. The mice were intravenously injected with 125I-labeled human Lp(a) or 125I-labeled human LDL in equimolar amounts and the lipoprotein allowed to circulate in vivo for 2 or 24 h. Transverse sections of the aortic root including sites of predilection for lesion formation at the commissures of the valve were prepared and examined after autoradiography. The autoradiographic grains over lesions and histologically uninvolved areas were enumerated and compared after normalization. Both Lp(a) and LDL demonstrated nearly ten times greater accumulation in lesions compared with histologically uninvolved areas from C57BL/6 mice. Analyses of histologically uninvolved areas from both strains of mice showed a significantly higher accumulation of Lp(a) than LDL. Finally, significantly higher accumulations of both Lp(a) and LDL occurred in the histologically uninvolved intima and subintima of lesion-prone C57BL/6 mice as compared with lesion-resistant C3H/HeJ mice after 5 weeks on the diets. We propose that enhanced accumulation of Lp(a) in the arterial wall accounts, in part, for the increased risk of cardiovascular disease.

Osteopenic mice: animal models of the aging skeleton.

While our understanding of the developmental biology of the skeleton, like that of virtually every other subject in biology, has been transformed by recent advances in human and mouse genetics, we still know very little, in molecular and genetic terms, about skeletal physiology. Thus, among the many questions that are largely unexplained are the following: why is osteoporosis mainly a women's disease? How is bone mass maintained nearly constant between the end of puberty and the arrest of gonadal functions? Molecular genetics has emerged as a powerful tool to study previously unexplored aspects of the physiology of the skeleton. Among mammals, mice are the most promising animals for this experimental work. This has been previously demonstrated e.g. through the tremendous impact of the different osteopetrotic models on our molecular understanding of osteoclastic bone resorption. Until recently the only way of studying bone loss situations and osteoporosis in mice was by using ovariectomy with all its limitations. Today, however, we have access to more sophisticated osteoporotic mouse-models from four different origins: Transgenic mice (HSV-TK), knock-out mice (OPG), inbred-strains (SAMP6), and through physiological modulation (icv application). These new models have already taught us several important lessons. The first is, that bone remodeling is more than just an autocrine/paracrine process. Multiple experimental evidence has demonstrated that the latter regulation exists, but genetics prove that there is no functional cross-control between resorption and formation. The second lesson is, that remodeling is, at least in part, subject to central regulation. Thus, osteoporosis is partly a central or hypothalamic disease. However, the most dramatic change and the most important advantage we feel is, that today we have models to test a new hypothesis regarding the etiology of osteoporosis before it turns to dogma. Taken together, mouse-studies may lead to a shift in our physiological understanding of skeleton biology and to the emergence of novel paradigms. These, in turn, should help us to devise new treatments for degenerative diseases of the skeleton such as osteoporosis and its associated clinical problems.

Transgenic and knock out mice in skeletal research. Towards a molecular understanding of the mammalian skeleton.

Our understanding of the biology of the skeleton, like that of virtually every other subject in biology, has been transformed by recent advances in human and mouse genetics. Among mammals, mice are the most promising animals for this experimental work. Because extensive genetic information exists, many mouse mutations are known, and cells from early mouse developmental stages are accessible, scientists have developed transgenic mice - mice in which a gene is introduced or ablated in the germ line. Thus far, we have analyzed more than 100 different transgenic and knock out models with various skeletal phenotypes, covering the major aspects of both skeletal development and skeletal maintenance. Based on these results we here present a first perspective on transgenic and gene knock out animals in skeletal research, including insights in signaling pathways controlling endochondral bone formation, in the regulation of osteoblast function, osteoclastic bone resorption and in bone tumorigenesis, as well as the central control of bone formation. The use of transgenic mice to dissect and analyze regulatory mechanisms in bone cell physiology and the pathogenesis of human bone diseases is an extremely powerful experimental tool. The data presented here demonstrate that the successful convergence of novel genetic approaches with the established and fundamental knowledge of bone biology has made a beginning.

Lipoprotein trafficking in vascular cells. Molecular Trojan horses and cellular saboteurs.

During the pathogenesis of atherosclerosis, inflammatory cells such as the monocyte-derived macrophage accumulate in the vessel wall where they release cytokines. Initially, cytokines may assist in CE removal of lipoprotein-derived cholesterol/CE hydrolysis to clear intracellular lipid. When plasma levels of LDL become elevated, the vessel wall becomes lipid-engorged over time because it is unable to traffick the large amounts of endocytosed LDL-CE from the cell. In addition, lipoprotein entrapment by the extracellular matrix can lead to the progressive oxidation of LDL because of the action of lipoxygenases, reactive oxygen species, peroxynitrite, and/or myeloperoxidase. A range of oxidized LDL species is thus generated, ultimately resulting in their delivery to vascular cells through several families of scavenger receptors (Fig 1). These molecular Trojan horses and cellular saboteurs once formed or deposited in the cell can contribute to, and participate in, formation of macrophage- and smooth muscle-derived foam cells. A lipid-enriched fatty streak along the vessel wall can ensue. In addition to foam cell development, products of LDL peroxidation may activate endothelial cells, increase smooth muscle mitogenesis, or induce apoptosis because of the effects of oxysterols and products of lipid peroxidation (Fig 1). Because antioxidant defenses may be limited in the microenvironment of the cell or within LDL, the oxidation process continues to progress. Enzymes associated with HDL such as PAF acetylhydrolase and paraoxonase can participate in the elimination of biologically active lipids, but diminished cellular antioxidant activity coupled with low levels of HDL may allow acceleration of the clinical course of vascular disease. There is still much to be learned about how modified LDL initiate cellular signals that lead to inflammation, mitosis, or cholesterol accumulation. The present challenges include elucidation of the key signaling events that regulate lipoprotein-derived cholesterol trafficking in the vessel wall, which can impact on the pathogenesis of vascular disease.

Nicotinamide adenine dinucleotide-specific glutamate dehydrogenase of Neurospora crassa. Isolation and sequences of several cyanogen bromide peptides from the NH2-terminal portion of the peptide chain.

Previous studies of the amino acid sequence of the NAD-specific glutamate dehydrogenase of Neurospora crassa (EC 1.4.1.2) resulted in the assignments of peptides to four fragments, the longest being the COOH-terminal 669 residues of the protein. A further study of peptides derived by cyanogen bromide cleavage by different separation methods has yielded additional peptides that have provided new information concerning the sequence and has given overlaps of previously known sequences. This has permitted establishment of 313 residues in one sequence (fragment II). This is in addition to a sequence of 43 residues (fragment I) at the NH2-terminal end and a sequence of 669 residues (fragment III) previously established at the COOH-terminal end of the molecule. The present status of our knowledge of the overall sequence is given in the accompanying papers, together with some views regarding the conformation of the protein (Haberland, M.E., Chen, C.-W., and Smith, E.L. (1980) J. Biol. Chem. 255, 7993-8000, and Austen, B.M., Haberland, M.E., and Smith, E.L. (1980) J. Biol. Chem. 255, 8001-8004).

Interaction of L-alpha-palmitoyl lysophosphatidylcholine with the AI polypeptide of high density lipoprotein.

The AI polypeptide chain from human high density serum lipoprotein has two accessible conformational states in aqueous solution. L-alpha-Palmitoyl lysophosphatidylcholine induces the transition between these two states at an equilibrium concentration of ligand of 2 X 10(-5)M, and the protein has a maximum binding capacity of 95 to 100 mol of lipid/mol of protein. The present study, together with previous investigations in this laboratory, suggests that the conformational state of AI in the presence of high levels of bound amphiphiles is similar to the in vivo state, and further, that this complex does not result from the insertion of AI into amphiphilic micelles. The mode of interaction of AI with amphiphilic ligands is shown to be significantly different from that of membrane proteins thus far investigated.

Self-association of cholesterol in aqueous solution.

Cholesterol has a maximum solubility in aqueous solutions of 1.8 mug/ml or 4.7 muM. It undergoes a thermodynamically reversible self-association with a critical micelle concentration of 25 to 40 nM at 25 degrees . The cholesterol micelle is heterogeneous in size, probably rodlike in shape, and stabilized by an unusually high interaction energy between the aggregated monomers.

Nicotinamide adenine dinucleotide-specific glutamate dehydrogenase of Neurospora crassa. IX. Isolation and sequences of several large cyanogen bromide peptides.

The isolation and sequences of several large peptides from cyanogen bromide cleavage of the 1030-residue polypeptide chain of the NAD-specific glutamate dehydrogenase of Neurospora crassa are described. One of these is in the 669-residue sequence of the COOH-terminal end of the protein. The remaining peptides have been aligned in two partial sequences in the NH2-terminal portion of the polypeptide chain.

Scavenger receptor-mediated recognition of maleyl bovine plasma albumin and the demaleylated protein in human monocyte macrophages.

Maleyl bovine plasma albumin competed on an equimolar basis with malondialdehyde low density lipoprotein (LDL) in suppressing the lysosomal hydrolysis of 125I-labeled malondialdehyde LDL mediated by the scavenger receptor of human monocyte macrophages. Maleyl bovine plasma albumin, in which 94% of the amino groups were modified, exhibited an anodic mobility in agarose electrophoresis 1.7 times that of the native protein. Incubation of maleyl bovine plasma albumin at pH 3.5 regenerated the free amino groups and restored the protein to the same electrophoretic mobility as native albumin. The demaleylated protein suppressed 75% of the hydrolysis of 125I-labeled malondialdehyde LDL and greater than 80% of 125I-labeled maleyl bovine plasma albumin. The ability of the demaleylated protein to compete was abolished after treatment with guanidine hydrochloride. Although ligands recognized by the scavenger receptor typically are anionic, we propose that addition of new negative charge achieved by maleylation, rather than directly forming the receptor binding site(s), induces conformational changes in albumin as a prerequisite to expression of the recognition domain(s). The altered conformation of the modified protein apparently persists after removal of the maleyl groups. We conclude that the primary sequence of albumin, rather than addition of new negative charge, provides the recognition determinant(s) essential for interaction of maleyl bovine plasma albumin with the scavenger receptor.

Role of lysines in mediating interaction of modified low density lipoproteins with the scavenger receptor of human monocyte macrophages.

The ability of the scavenger receptor of human monocyte macrophages to recognize human low density lipoproteins (LDL) progressively modified by three lysine-specific reagents, malondialdehyde, acetic anhydride, or succinic anhydride, has been investigated. Regardless of the reagent utilized, receptor-mediated uptake was dependent upon modification of greater than 16% of the peptidyl lysines rather than upon the net negative charge of derivatized LDL. Rates of lysosomal hydrolysis of acetyl-LDL and succinyl-LDL increased as a function of progressive modification and reflected the amount of derivatized LDL binding to the receptor. Succinylation or acetylation of greater than 60% of the lysines was necessary to attain maximal ligand binding, internalization, and degradation. In contrast, modification of only 16% of the peptidyl lysines by malondialdehyde resulted in maximal levels of binding, uptake, and hydrolysis. The expression of receptor recognition site(s) appears to depend upon the charge modification of critical lysine residues of the LDL protein rather than the net negative charge of the lipoprotein complex. Malondialdehyde, a bifunctional reactant, may modify surface and sequestered lysines concomitantly and thus promote efficient formation of the recognition site(s).