The role of signaling pathways in osteoblast gravity perception

Bone loss is one of the major problems in long term spaceflight. This physiological consequence of microgravity is the rapid loss of weightbearing bone that is associated with skeletal unloading. Moreover, we have previously noted that sera deprived osteoblasts do not have a normal response to sera in microgravity. Where exercise (mechanical loading) has been shown to increase bone formation and stimulate osteoblastic function, the mechanisms underlying signal transduction of mechano-perception is yet to be fully understood. Osteoblasts have been shown to respond to mechanical stress such as fluid shear, bending, flexing and compression. The type of stress and amount of stress determine the osteoblast response Recently we have discovered that the isolated osteoblast responds to a very short pulse of g-force compression. The possible regulatory sensors include mechano-sensitive calcium channels, autrocrine responses to stress, response to FAK/integrin, alterations in the cytoskeleton as well as other known growth factor and cytokine receptors. The secondary signal may include growth factor related kinases such as ERK, p38 and JNK map kinase (MAPK) pathways. Experimental evidence suggests that normal osteoblast response to stress and sera requires normal earth gravity.     

Electron microscope observations on chemo- and mechano-receptor cells of fishes

Summary This paper reports on the fine structure of chemo and mechano-receptor cells found in three species of fishes (Corydoras paleatus, Cnesterodon decemmaculatus, Fitzroyia lineata).Taste cells were studied in the food-finding barbels of adult species belonging to the Genus Corydoras. They are characterized by the presence of a great amount of vesicular material concentrated at the level of the apical and medial region. Most of these cells terminate at the barbel surface by means of a cylindrical or tapered extremity devoid of sensory hairs. It was possible to observe, in some cases, the existence of short and ill defined microvilli. The basal pole of each sensory cell contacts with several sensory nerve fibers. These fibers contain mitochondria and a few vesicles.The fine structure of the olfactory neurons was studied in full-developed embryos of Cnesterodon and Fitzroyia. The olfactory sensory hairs consist of long cilia which project into the lumen of the olfactory pit. Cilia arise from the olfactory knob which is merely an apical swelling of the dendrite. The dendrite of the olfactory neuron shows profiles of small tubules, aligned parallel to its length. Near the basement membrane of the epithelium groups of axons are seen encased in the surface of the sustentacular cells.The mechano-receptor cells studied were: 1.) The sensory cells existing in the neuromasts of the lateral line system of Cnesterodon and Fitzroyia, and 2.) the receptor cells of the ampullar crests of the same species.Neuromast receptor-cells have well developed sensory hairs which consist of cilia and microvilli. It is highly probable that each receptor cell, like those of the vestibular epithelium, bears only one cilium asymmetrically located in relation to the units of the sensory process. One of the most striking characteristics of this type of cell is the existence of a high amount of vesicular material accumulated in the cytoplasm of the basal region; it is at this level that the nerve fibers take contact with the receptor cell membrane.Three main types of neuroepithelial junction are described in the neuromasts (nerve fiber deeply recessed in the cytoplasm, calyx type and knob-like ending). In these junctions the vesicular material is almost exclusively concentrated in the cytoplasm of the receptor cell, while only few vesicles are seen within the neuroplasm of the sensory fibers.The receptor cells occuring in the ampullar crests of Cnesterodon and Fitzroyia show many structural characteristics similar to those present in neuromasts' receptor cells. Like these, they bear sensory hairs consisting of several microvilli and only one cilium which is always asymmetrically located within the group of hairs. The basal region of the cell is filled with a large amount of small vesicles. Nerve endings also show vesicles but they are less in number than inside the cytoplasm of the receptor cell.Comments are made on the apparent significance of the sensory hairs. These structures are considered (in chemo-receptor cells) as devices serving to enlarge the active surface of the cell and increasing by this way the effectiveness of the whole receptive system. In mechano-receptor cells cilia and microvilli may act as levers of different mechanical characteristics which convey stimuli to the receptor-cell cytoplasm.In this paper three main types of neuroepithelial junctions connecting receptor cells with the central nervous system are described.     

A theoretical approach to understanding of the vestibular perception organization in microgravity condition

This paper should be viewed as a part of our attempts to arrive at a quantitative understanding of some contradictory experimental phenomena in the vestibular perception. The most popular remains the Steinhausen's model of perception, in which the endolymph circulation, caused by the angular acceleration, is "measured" by the displacement of cupulae diaphragm. Though displacements of the cupulae top were experimentally observed, the thorough mathematical analysis shows that the applicated stop-stimuli were too much over the range of adequate stimulation conditions, so the cupulae was teared off from its normal position. The more natural mechanism of perception was proposed by McLaren & Hillman. It was experimentally demonstrated that the cupulae diaphragm remained margin attached during the normal stimulation conditions only with its bottom moved according to the value of the applicated angular acceleration. A question only arises, how will be the hair cells excited by low level stimulation, when elastic deformations of the cupulae are small and there is no visible shift of the cupulae bottom? The response is to find in the third mechanism formulated by Schmaltz, which connected the excitation of hair cells with the process of endolymph diffusion through the cupulae towards the subcupulae space.     

Geometry, thermodynamics, and protein

We derive a new continuous free energy formula for protein folding. We obtain the formula first by adding hydrophobic effect to a classical free energy formula for cavities in water. We then obtain the same formula by geometrically pursuing the structure that fits best the well-known global geometric features of native structures of globular proteins: 1. high density; 2. small surface area; 3. hydrophobic core; 4. forming domains for long polypeptide chains. Conformations of a protein are presented as an all atom CPK model where each atom is a ball B(x_i,r_i). All conformations satisfy generally defined steric conditions. For each conformation P of a globular protein, there is a closed thermodynamic system Ω_P⊃P bounded by the molecular surface M_P. Both methods derive the same free energy aV(P)+bA(P)+cW(P), where a,b,c>0, V(P), A(P), and W(P) are volume of Ω_P, area of M_P, and area of the hydrophobic surface W_P⊂M_P, which quantifies hydrophobic effect.Minimizing W(P) is sufficient to produce statistically significant native like secondary structures and hydrogen bonds in the proteins we simulated.     

The thermostatics and thermodynamics of cotransport

The thermostatics of cotransport are reviewed. A static-head equilibrium state across a cotransport system, without leaks, is thought to occur when the electrochemical potential of the driven solute, B prevents net flow of the driving solute, A. For a symport this gives the relationship (formula: see text) Where n is the stoichiometric coefficient, namely the number of moles of A transported per mole of B. (2) If either a symporter with a 2:1 stoichiometric coefficient and a 1:1 symporter, or alternatively, a 1:1 symporter and a 1:1 antiporter are placed in a series membrane array, then the predicted static-head equilibrium across the entire array conflicts with the zeroth law of thermodynamics. (3) There are two major reasons for this failure of cotransport theory; these are: (A) the thermostatic relationships derived shown in Point 1 are based on the assumption that the cotransport process takes place within a closed system. However, the membrane and the external reservoirs are open to the cotransported ligands. It follows that A and B in the external reservoirs can vary independently of the changes within the cotransport process. As no chemical reaction between A and B occurs in the external solutions, reactions within the membrane phase do not affect the equilibrium between the transported ligands in the open reservoirs. (B) It is assumed that the law of mass action can be applied to the cotransport chemical reactions within the membrane phase, without any allowance for the fact that these reactions occur within a 'small thermodynamic system'. Any proper analysis of the chemical potential of the transported intermediate must consider the effects of lower order ligand-carrier forms, which coexist and compete for space with the higher order cotransported forms on the binding matrix. If account is taken of this necessity, then a simple extension of the work of Hill and Kedem (1966) J. Theor. Biol. 10, 399-441 shows that: (a) the static-head equilibrium state cannot exist; (b) the stoichiometry of cotransport, whether symport, or antiport, does not affect the static-head distribution of cotransported ligands; (c) the hypothetical net charge of the transported ligand-carrier complex does not affect static-head equilibrium; (d) the only equilibrium state where there is zero net flow of both driving and driven transported ligand is at true equilibrium when the ligands are uniformly distributed across the membrane. (4) It is deduced that cotransport is not entirely an affinity-driven, but is partially an entropy-driven process.(ABSTRACT TRUNCATED AT 400 WORDS)     

Neural and neuroendocrine adaptations to microgravity and ground-based models of microgravity

The functional properties of the motor system of humans and non-human primates are readily responsive to microgravity. There is a growing body of evidence that significant adaptations occur in the spinal cord and muscle in response to prolonged exposure to microgravity. Further, there is evidence that the processing of sensory information from the periphery, particularly that input associated with the function of muscle tendons and joints, is significantly altered as a result of prolonged microgravity. We present evidence that the fundamental neural mechanisms that control the relative activity of the motor pools of a slow and fast extensor muscle is changed such that a slow, postural muscle is less readily activated during locomotion following spaceflight. Another type of change observed in mammals exposed to spaceflight relates to the release of a growth factor, called bioassayable growth hormone, which is thought to be released from the pituitary. When an individual generates a series of isometric plantarflexor contractions, the plasma levels of bioassayable growth hormone increases significantly. This response is suppressed after several days of continuous bedrest or spaceflight. These results suggest a unique neuroendocrine control system and demonstrate its sensitivity to chronic patterns of proprioceptive input associated with load-bearing locomotion.     

Effects of transient PTH on early proliferation, apoptosis, and subsequent differentiation of osteoblast in primary osteoblast cultures

In primary calvarial osteoblast cultures derived from transgenic mice expressing green fluorescent protein (GFP) under the control of 3.6-kb Col1a1 promoter, the emergence of GFP signal marks the transition of multipotential osteoprogenitors into preosteoblasts. Early transient treatment (days 1-7) of these cultures with parathyroid hormone (PTH) has an anabolic effect that is not associated with an increase in total DNA content or cell number in day 21 cultures. In the present study, the effect of early PTH treatment on cell proliferation and apoptosis was examined in greater detail in GFP(+) and GFP(-) cells using flow cytometry. In preconfluent cultures, PTH significantly reduced the proportion of cells in S phase but increased those in G(0)/G(1) and G(2)+M phases in both GFP(+) and GFP(-) subpopulations. PTH decreased apoptosis only in GFP(-) but not GFP(+) cells, indicating an increased survival of GFP(-) cells. In contrast, PTH did not change the amounts of cell proliferation and apoptosis seen in either compartment after these cultures reached confluence. To further assess the effect of early PTH treatment on osteogenic differentiation, secondary cultures of sorted GFP(+) or GFP(-) cells were obtained from day 7 primary cultures that had been treated for 1 wk with PTH. This treatment resulted in larger areas of GFP expression accompanied by increased xylenol orange/von Kossa staining in the secondary cultures of GFP fractions. Early transient PTH treatment appears to enhance the commitment of progenitor cells to an osteogenic fate and results in a higher proportion of cells that achieve full osteoblast differentiation.     

The thermodynamics of thiol sulfenylation

Protein sulfenic acids are essential cysteine oxidations in cellular signaling pathways. The thermodynamics that drive protein sulfenylation are not entirely clear. Experimentally, sulfenic acid reduction potentials are hard to measure, because of their highly reactive nature. We designed a calculation method, the reduction potentials from electronic energies (REE) method, to give for the first time insight into the thermodynamic aspects of protein sulfenylation. The REE method is based on the correlation between reaction path-independent reaction energies and free energies of a series of analogous reactions. For human peroxiredoxin (Tpx-B), an antioxidant enzyme that forms a sulfenic acid on one of its active-site cysteines during reactive oxygen scavenging, we found that the reduction potential depends on the composition of the active site and on the protonation state of the cysteine. Interaction with polar residues directs the RSO(-)/RS(-) reduction to a lower potential than the RSOH/RSH reduction. A conserved arginine that thermodynamically favors the sulfenylation reaction might be a good candidate to favor the reaction kinetics. The REE method is not limited to thiol sulfenylation, but can be broadly applied to understand protein redox biology in general.     

The thermodynamics of water-protein interactions

Information about the effects of water on protein structure and function can be obtained from studies on freeze dried protein powders of varying water content. Sorption isotherms of water on proteins can be used to obtain thermodynamic quantities for water-protein interactions. Since such isotherms show hysteresis, there is doubt in regard to their interpretation.General expressions for the thermodynamic quantities of sorption are derived. If isotherms represent data at equilibrium, it is possible to calculate these thermodynamic quantities.There are two types of hysteresis, non-equilibrium hysteresis and equilibrium hysteresis. Absorption and desorption isotherms can show equilibrium hysteresis if different protein conformations, which are only slowly interconvertible, can be present. In this case valid thermodynamic quantities can be obtained. Experimental tests for equilibrium hysteresis are presented. More experiments are needed before definite conclusions can be drawn in regard to isotherms in the literature.If the protein conformation in a protein powder is similar to the protein conformation in aqueous solution, equilibrium data obtained from sorption isotherms can be used to approximate thermodynamic quantities for the interaction of water with proteins in aqueous solution. Examination of what experimental evidence is available indicates that the protein in powders prepared by desorption of water should have a conformation similar to that in solution. Further study of such samples will help to clarify the thermodynamics of water-protein interactions in aqueous solution.     

The thermodynamics of general anesthesia

It is known that the action of general anesthetics is proportional to their partition coefficient in lipid membranes (Meyer-Overton rule). This solubility is, however, directly related to the depression of the temperature of the melting transition found close to body temperature in biomembranes. We propose a thermodynamic extension of the Meyer-Overton rule, which is based on free energy changes in the system and thus automatically incorporates the effects of melting point depression. This model accounts for the pressure reversal of anesthesia in a quantitative manner. Further, it explains why inflammation and the addition of divalent cations reduce the effectiveness of anesthesia.     

The retinoblastoma protein in osteoblast differentiation and osteosarcoma

Osteogenic sarcoma (osteosarcoma) is the most common primary tumor of bone. It accounts for approximately 19% of all malignant tumors of the bone. Of all the molecular targets altered during the genesis of osteosarcoma, the retinoblastoma gene (RB1) shows the highest frequency of inactivation. Published data from human osteosarcoma tumors and in vivo and in vitro model systems support a role for the retinoblastoma gene family in bone development and tumorigenesis. Although a variety of bone tumors, depending on the cell of origin, including osteoclasts or osteoclast-like cells, chondroblasts, and fibroblasts, are described, for the purpose of this review we will focus primarily on the tumors arising from the osteoblast lineage.     

The thermodynamics of solvent exchange

A model for solvation in mixed solvents, which was developed for the free energy and preferential interaction [J. A. Schellman (1987), Biopolymers, Vol. 26, pp. 549–559; (1990), Biophysical Chemistry, Vol. 37, pp. 121–140; (1993), Biophysical Chemistry, Vol. 45, pp. 273–279], is extended in this paper to cover the thermal properties: enthalpy, entropy, and heat capacity. An important result is that the enthalpy of solvation H? responds directly to the fraction of site occupation. This differs from the free energy ? and preferential interaction Γ₃₂, which are measures of the excess binding above a random distribution of solvent molecules. In other words, the enthalpy is governed by K while ? and Γ₃₂ are governed by (K ? 1) where K is the equilibrium constant on a mole fraction scale [Schellman (1987)]. The solvation heat capacity C?p consists of two term: (1) the intrinsic heat capacity of species in solution with no change in composition, and (2) a term that accounts for the change in composition that accompanies solvent exchange. Binding to biological macromolecules is heterogeneous but experiementalists must use binding isotherms that assume the homogeneity of sites. Equations are developed for the interpretation of the experimental parameters (number of sites n_exp, equilibrium constant K_exp, and enthalpy, Δh_exp), when homogeneous formulas are applied to the heterogeneous case. It is shown that the experimental parameters for the occupation and enthalpy are simple functions of the moments of the distribution of equilibrium constants over the sites. In general, n_exp is greater than the true number of sites and K_exp is greater than the average of the equilibrium constants. The free energy and preferential interaction can be fit to a homogenious formula, but the parameters of the curve are not easily represented in terms of the moments of distributions over the sites. The strengths and deficiencies of this type of thermodynamic model are discussed. © 1994 John Wiley & Sons, Inc.     

Control, regulation and thermodynamics of free-energy transduction

The quantitative formalism called Metabolic Control Theory makes it possible to be precise in discussions of metabolic control. To illustrate this, I will mention 2 experimental systems where free energy is converted from one form to another, i.e., bacteriorhodopsin liposomes and mitochondrial oxidative phosphorylation. More specifically I shall discuss how the distribution of the control of fluxes, concentrations and potentials, among the various enzymes (catalysts) in these systems has been measured and how this distribution can be understood in terms of the enzyme properties. From the outset, Metabolic Control Theory was valid for branched metabolic pathways with non-linear kinetics. Yet, it seemed to be limited to metabolic pathways without enzyme-enzyme interactions and to steady states. It is now clear that these limitations were apparent only and recent extensions to Metabolic Control Theory deal explicitly with enzyme-enzyme interaction and with transient-time analysis. Other limitations are inherent. For instance, Metabolic Control Theory pays for its clarity and exactness by being limited to small modulations. Mosaic Non Equilibrium Thermodynamics and Biochemical System Analysis are formalisms that deal with larger changes, at the cost of accuracy and exactness.     

Lipid bilayers: thermodynamics, structure, fluctuations, and interactions

This article, adapted from our acceptance speech of the Avanti Award in Lipids at the 47th Biophysical Society meeting in San Antonio, 2003, summarizes over 30 years of research in the area of lipid bilayers. Beginning with a theoretical model of the phase transition (J.F.N.), we have proceeded experimentally using dilatometry and density centrifugation to study volume, differential scanning calorimetry to study heat capacity, and X-ray scattering techniques to study structure of lipid bilayers as a function of temperature. Electron density profiles of the gel and ripple phases have been obtained as well as profiles from several fluid phase lipids, which lead to many structural results that compliment molecular dynamics simulations from other groups. Using the theory of liquid crystallography plus oriented lipid samples, we are the first group to obtain both material parameters (KC and B) associated with the fluctuations in fluid phase lipids. This allows us to use fully hydrated lipid samples, as in vivo, to obtain the structure.     

Bacterial chemotaxis: information processing,thermodynamics, and behavior

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Redistribution of bodily fluids under conditions of microgravity and in microgravity models

This review is focused on the redistribution of blood and other bodily fluids along the body axis in the cranial direction under conditions of microgravity or during simulation of the physiological effects of microgravity. This redistribution of bodily fluids in the direction of the thorax or head results in respective physiological responses and induces a whole cascade of secondary adaptation mechanisms. Changes in central venous pressure, heart cavity volume, kidney functioning, and hormonal volume regulation lead to adaptive modifications in bodily fluid sectors. Modification of the hemodynamic in the splanchnic vascular system influences the organs of the abdominal cavity. Pharmacological correction accelerates the adaptation of the human body to unusual living conditions.