Tartrate-resistant acid ATPase as a cytochemical marker for osteoclasts

We present a modified histochemical method for staining osteoclasts and adjacent mononuclear cells which takes advantage of the recently described substrate specificity for ATP of osteoclastic acid phosphatase. Staining of osteoclasts using ATP as substrate exhibits by light microscopy the same tartrate resistance as conventional acidic phosphatases, without the bone surface staining seen with other substrates. This feature, coupled with specific staining of fewer vicinal mononuclear cells, makes this method potentially useful for studying osteoclast ontogeny and function.     

Persistence of an amine uptake system in cultured rat sympathetic neurons which use acetylcholine as their transmitter

Cultures of dissociated rat superior cervical ganglion neurons (SCGN) were treated with the sympatholytic agent, guanethidine. When treated within the first couple of weeks in vitro, the neurons were rapidly destroyed. The cells grew less susceptible to the toxic effects of guanethidine with age in vitro. Moreover, the apparent affinity, Km, of the transport molecule for norepinephrine (NE) and guanethidine remained essentially unchanged between 2 and 7 wk in culture, as did the maximum velocity of transport (Vmax). This is at a time when previous studies have shown these neurons to be using acetylcholine (ACh) as their neurotransmitter. Cultures which were grown without supporting cells and from which cholinergic synaptic interactions were recorded physiologically were processed for autoradiography after incubation with [3H]NE. All cell bodies and processes seen had silver grains accumulated over them. These experiments show that sympathetic neurons in vitro maintain their amine uptake system relatively unchanged, even though they use ACh as their transmitter. The implications of these findings are discussed.     

Regulation of podosomes by intracellular pH in avian osteoclasts

The effects of changes in intracellular pH (pH1) on the organization of the clear zone of isolated avian osteoclasts in culture were studied. The distribution of podosomes, the close contact areas that mediate the adhesion of osteoclasts to the substrate, was investigated by decoration of microfilaments with fluorescent phalloidin. Intracellular acidification by butyric acid induces significant increase of podosome formation at the level of the clear zone compared to controls. Conversely, alkalinization by HCO3- reduces the percentage of osteoclasts with podosomes. A role of pH1 on the adhesion of the osteoclasts to the substrate is hypothesized.     

Single-channel currents activated by low intracellular pH in cultured hippocampal neurons of rat.

Patch clamp technique was applied to the plasma membrane of cultured hippocampal neurons of rat. Elementary currents of a cation-selective channel were elicited by low intracellular pH (pHi 3.5-4.5). Channel activity starts with 1-2 min delay from the application of low pHi, and persists upon restoration of physiological pH conditions. The channel has a conductance of approx. 110 pS in symmetrical 300 mM NaCl, and is strongly selective for cations over anions. The channel is active over the whole voltage range tested (from +75 mV to -75 mV). Mean open time is function of voltage, increasing with depolarization. Low pH applied extracellularly did not activate the channel.     

Visualization of bioluminescence as a marker of gene expression in rhizobium-infected soybean root nodules

The linked structural genes lux A and lux B, encoding bacterial luciferase of a marine bacterium Vibrio harveyi, were fused with the nitrogenase nifD promoter from Bradyrhizobium japonicum and with the P1 promoter of pBR322. Both fusions were integrated into the B. japonicum chromosome by site-specific recombination. Soybean roots infected with the two types of rhizobium transconjugants formed nitrogen-fixing nodules that produced bright blue-green light. Cells containing the P1 promoter/lux AB fusion resulted in continuously expressed bioluminescence in both free-living rhizobium and in nodule bacteriods. However, when under control of the nifD promoter, luciferase activity was found only in introgen-fixing nodules. Light emission from bacteroids allowed us to visualize and to photograph nodules expressing this marker gene fusion in vivo at various levels of resolution, including within single, living plant cells. Localization of host cells containing nitrogen-fixing bacteroids within nodule tissue was accomplished using low-light video microscopy aided by realtime image processing techniques developed specifically to enhance extreme low-level luminescent images.     

The use of titanium dioxide as an inert marker for digestion studies in raptors.

1. TiO2 is not a suitable marker for digestibility studies involving raptors. 2. Complete recovery of the 'inert' marker was not achieved. 3. Total collection of faeces is a more accurate method to determine food absorption. 4. Freezing sample solutions prior to colorimetric analysis can produce misleading results. 5. The percentage food absorbed from the intestine in Falconiformes is about 85% by dry weight.     

Modulation of TRPM2 by acidic pH and the underlying mechanisms for pH sensitivity

TRPM2 is a Ca²⁺-permeable nonselective cation channel that plays important roles in oxidative stress–mediated cell death and inflammation processes. However, how TRPM2 is regulated under physiological and pathological conditions is not fully understood. Here, we report that both intracellular and extracellular protons block TRPM2 by inhibiting channel gating. We demonstrate that external protons block TRPM2 with an IC₅₀ of pH_o = 5.3, whereas internal protons inhibit TRPM2 with an IC₅₀ of pH_i = 6.7. Extracellular protons inhibit TRPM2 by decreasing single-channel conductance. We identify three titratable residues, H958, D964, and E994, at the outer vestibule of the channel pore that are responsible for pH_o sensitivity. Mutations of these residues reduce single-channel conductance, decrease external Ca²⁺ ([Ca²⁺]_o) affinity, and inhibit [Ca²⁺]_o-mediated TRPM2 gating. These results support the following model: titration of H958, D964, and E994 by external protons inhibits TRPM2 gating by causing conformation change of the channel, and/or by decreasing local Ca²⁺ concentration at the outer vestibule, therefore reducing [Ca²⁺]_o permeation and inhibiting [Ca²⁺]_o-mediated TRPM2 gating. We find that intracellular protons inhibit TRPM2 by inducing channel closure without changing channel conductance. We identify that D933 located at the C terminus of the S4-S5 linker is responsible for intracellular pH sensitivity. Replacement of Asp⁹³³ by Asn⁹³³ changes the IC₅₀ from pH_i = 6.7 to pH_i = 5.5. Moreover, substitution of Asp⁹³³ with various residues produces marked changes in proton sensitivity, intracellular ADP ribose/Ca²⁺ sensitivity, and gating profiles of TRPM2. These results indicate that D933 is not only essential for intracellular pH sensitivity, but it is also crucial for TRPM2 channel gating. Collectively, our findings provide a novel mechanism for TRPM2 modulation as well as molecular determinants for pH regulation of TRPM2. Inhibition of TRPM2 by acidic pH may represent an endogenous mechanism governing TRPM2 gating and its physiological/pathological functions.     

Copper bioavailability and rhizosphere pH changes as affected by nitrogen supply for tomato and oilseed rape cropped on an acidic and a calcareous soil

Vineyard soils have been contaminated by long-term applications of copper salts as fungicides against mildew, raising the question of the bioavailability (and toxicity) of such accumulated Cu to cultivated plants which can replace vines. The aim of this study was to assess, in an acidic and a calcareous Cu-contaminated soil, how the extractability and bioavailability of soil Cu was affected by pH changes in the rhizosphere of two plant species (oilseed rape and tomato), in response to various forms of nitrogen supply (nitrate only or both nitrate and ammonium). Besides shoot analysis, the experimental approach used in the present work provided an easy access to both roots and rhizosphere soil. Roots of tomato and rape induced a systematic acidification in the calcareous soil while root-induced alkalinization occurred in the acidic soil. Whilst few differences were found between treatments in the calcareous soil, oilseed rape took up more Cu and also alkalinized its rhizosphere more strongly than tomato in the acidic soil. The growth of tomato roots was restricted in the acidic soil, while that of oilseed rape was not, suggesting that tomato was either more sensitive to soil acidity and/or Cu toxicity. A major finding was that, in the acidic soil, Cu bioavailability increased with increasing rhizosphere pH. This was largely due to the enhanced accumulation of Cu in the root compartment of both species with increasing rhizosphere pH. The hypothetical explanation proposed here is that Cu binding to root cell walls played a major role in the accumulation of Cu into the plant. Apoplasmic Cu (Cu bound to cell walls) would indeed be expected to increase with increasing pH as a consequence of the pH-dependency of the charges of cell wall constituents.     

Wheat germ agglutinin-gold as a novel marker for mesectoderm formation in mouse embryos cultured in vitro

The routes of movement of mesectoderm cells in mammalian embryos have not yet been investigated experimentally due to technical problems. However, the recent development of in vitro culture methods have made an experimental approach to this problem in mouse and rat embryos possible. We have used combined lectin and colloidal-gold (WGA-Au) probe as a nontraumatic, easily detectable mesectoderm marker. The probe is introduced into the amniotic cavity by microinjection. All of the cells lining the cavity, including the mesectoderm precursors, phagocytose the colloidal gold, which is then stored in membrane-bound vesicles. The probe remains inside the target mesectoderm cells after their migration into the mesoderm compartment. Vesicles containing gold are detectable in both ultrathin and semithin sections. The applicability of WGA-HRP as a probe was also assessed because of the many properties it shares with WGA-Au, but it proved to be unsatisfactory for this purpose because it is transferred between cells and also to the extracellular spaces.     

Aldolase of lactic Acid bacteria: a case history in the use of an enzyme as an evolutionary marker

[This corrects the article on p. 453 in vol. 37.].     

Synthesis and 31P NMR characterization of new low toxic highly sensitive pH probes designed for in vivo acidic pH studies

With the aim to provide sensitive 31P NMR probes of intra- and extracellular pH gradients that may reach cellular acidic compartments in biological systems, new alpha-aminophosphonates were designed to meet basic requirements such as a low pK(a)s and a great chemical difference (Deltadelta(ab)) between the limiting 31P NMR chemical shifts in acidic (delta(a)) and basic (delta(b)) media. A series of six phosphorylated pyrrolidines and linear aminophosphonates were synthesized using aminophosphorylation reactions and were screened for cytotoxicity on cultured Müller cells. Among the compounds not being toxic under these conditions, three molecules were selected since they displayed the best in vitro (in several phosphate buffers and in a cytosol-like solution) properties as 31P NMR acidic pH markers, that is 3, 5 and 9, having the pK(a) values of 3.63, 5.89 and 5.66, respectively. The Deltadelta(ab) values of these pH markers were at least 3 times larger than that of standard 31P NMR probes, with a low sensitivity to ionic strength changes. From these data, it was proposed that 3, 5 and 9 could be used as reporting probes of subtle proton movements in acidic compartments, an area that still remains poorly investigated using non invasive 31P NMR methods.     

Elemental concentrations in plant tissues as influenced by low pH soils

Summary Soils influenced by acid mine drainage (pH<5.0) are characterized by low concentrations of essential nutrients and increased solubility of heavy metals. The conditions typically reduce plant establishment and growth. However, river birch (Betula nigra L.) is commonly found along low pH streams in southeastern Ohio. The objective of this study was to determine the concentration of Al, Mn, Ca and Mg inB. nigra tissues.The results indicate Al and Mn are accumulating inB. nigra when compared to other species. Within river birch, Al concentrations are highest in roots; Mn concentrations are highest in leaves. There is not a concomitant reduction in Ca and Mg concentrations as suggested by soil levels.     

The use of polymer fibers as a substrate for cultured nerve tissue

A study was made of the reaction of cells to administration of polymeric fibers containing amino acids--phenylalanine and leucine--to the culture of nerve tissue. The data obtained have shown that polymeric fibers are some acceptable substrate for the migration of astrocytes and oligodendrocytes and for the growth of nerve cell axons.     

Identification of an anti-aldolase autoantibody as a diagnostic marker for diabetic retinopathy by immunoproteomic analysis

Circulating autoantibodies specific for retinal proteins are associated with retinal destruction in patients with diabetic retinopathy (DR). In this study, we screened diabetic sera for the presence of anti-retinal autoantibodies with an aim of developing diagnostic markers for DR. Immunoblot analysis of DR patients' sera with human retinal cytosolic proteins revealed a higher incidence of anti-retinal autoantibodies, compared to normal blood donors or diabetic patients without DR. Anti-retinal protein autoantibody profiles of DR patient sera were obtained by 2-DE immunoblot analysis. Specifically, 20 protein spots reactive with DR patient sera were identified by ESI-MS/MS. Of these spots, 14 were specific for DR patients, and 4 reacted with both non-proliferative DR (non-PDR) and PDR sera. The anti-aldolase autoantibody was selected as a DR marker candidate, and specific reactivity of DR patient sera was confirmed by immunoblot analysis with rabbit aldolase. The serum anti-aldolase autoantibody level was measured by ELISA. DR patients showed significantly higher autoantibody levels than normal donors or diabetic patients without retinopathy. However, no significant differences were observed between non-PDR and PDR patients, suggesting that the level of anti-aldolase autoantibody is not determined by the severity of retinopathy in diabetic patients. Our data collectively demonstrate that the anti-aldolase autoantibody serves as a useful marker for DR diagnosis.     

Glyceraldehyde-3-phosphate dehydrogenase as a biochemical marker of cytotoxicity by vinyl sulfones in cultured murine spleen lymphocytes

Recently, vinyl sulfones have been observed to selectively inhibit glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which is an important ATP-generating enzyme in glycolysis. The possibility of using GAPDH as a biochemical parameter of cytotoxicity by vinyl sulfones was investigated using mouse lymphocytes. Incubation of lymphocyte GAPDH with ethylvinyl sulfone resulted in a pseudo-first-order loss of enzyme activity. The exposure of lymphocytes to ethylvinyl sulfone resulted in the decrease of GAPDH activity followed by ATP depletion and cell death, which were both dependent on the concentration of ethylvinyl sulfone. A further study on the time-dependent change indicated that cell death was preceded by ATP loss. Compared to ethylvinyl sulfone, divinyl sulfone was more than 8 times more potent in causing either ATP depletion or cell death.Abbreviations DTT dithiothreitol - GAPDH glyceraldehyde-3-phosphate dehydrogenase - NAD nicotinamide adenine dinucleotide     

Oligomeric rearrangement of tick-borne encephalitis virus envelope proteins induced by an acidic pH.

The flavivirus envelope protein E undergoes irreversible conformational changes at a mildly acidic pH which are believed to be necessary for membrane fusion in endosomes. In this study we used a combination of chemical cross-linking and sedimentation analysis to show that the envelope proteins of the flavivirus tick-borne encephalitis virus also change their oligomeric structure when exposed to a mildly acidic environment. Under neutral or slightly alkaline conditions, protein E on the surface of native virions exists as a homodimer which can be isolated by solubilization with the nonionic detergent Triton X-100. Solubilization with the same detergent after pretreatment at an acidic pH, however, yielded homotrimers rather than homodimers, suggesting that exposure to an acidic pH had induced a simultaneous weakening of dimeric contacts and a strengthening of trimeric ones. The pH threshold for the dimer-to-trimer transition was found to be 6.5. Because the pH dependence of this transition parallels that of previously observed changes in the conformation and hydrophobicity of protein E and that of virus-induced membrane fusion, it appears likely that the mechanism of fusion with endosomal membranes involves a specific rearrangement of the proteins in the viral envelope. Immature virions in which protein E is associated with the uncleaved precursor (prM) of the membrane protein M did not undergo a low-pH-induced rearrangement. This is consistent with a protective role of protein prM for protein E during intracellular transport of immature virions through acidic compartments of the trans-Golgi network.