Search

A high-throughput microfabricated all-glass microchip, lipid biochip, was created and used to measure fluorescently tagged antibody binding to dinitrophenol (DNP) haptens in planar supported phospholipid/cholesterol lipid bilayers as a function of cholesterol-to-lipid molar ratio (XCHOL). Multiple parallel microchannels etched in the lipid biochip allowed simultaneous measurement of antibody binding to hapten-containing and hapten-free lipid bilayers, for a range of aqueous antibody concentrations. Specific and nonspecific antibody binding to the supported lipid bilayers was determined from the internally calibrated intensity of the surface fluorescence using total internal reflectance fluorescence (TIRF) microscopy. The TIRF intensity data of the specific antibody binding were fitted to the Langmuir isotherm and Hill equation models to determine the apparent dissociation constant Kd, the maximum fluorescence parameter F∞, and binding cooperativity n. As XCHOL increased from 0 to 0.50, Kd exhibited a minimum of ~ 4 μM and n reached a maximum of ~2.2 at XCHOL ≈ 0.20. However, F∞ appeared to be insensitive to the cholesterol content. The nonspecific binding fraction (NS), defined as the ratio of the TIRF intensity for hapten-free bilayers to that with hapten, showed a minimum of ~0.08 also at XCHOL ≈ 0.20. The results suggest that cholesterol regulates the specific binding affinity and cooperativity, as well as suppresses nonspecific binding of aqueous antibody to a planar supported lipid bilayer surface at an optimal cholesterol content of XCHOL ≈ 0.20. Interestingly, for XCHOL ≈ 0.40, NS reached a maximum of ~0.57, suggesting significant packing defects in the lipid bilayer surface, possibly as a result of lipid domain formation as predicted by the lipid superlattice model. We conclude that cholesterol plays a significant role in regulating both specific and nonspecific antibody/antigen binding events on the lipid bilayer surface and that our lipid biochip represents a new and useful high-resolution microfluidic device for measuring lipid/protein surface binding activities in a parallel and high-throughput fashion.

The mechanism behind the distinctive non-Michaelis-Menten, bell-shaped kinetics of cAMP accumulation in brown adipocytes (which underlies the similar kinetics of UCP1 and beta(1)-adrenoreceptor gene expression) was investigated. A theoretical dual component analysis indicated that the observed dose-response curves could be constructed as the resultant of a stimulatory and an inhibitory component. Experimentally, inhibition of the alpha(1)-component of the norepinephrine response revealed the underlying existence of a much larger stimulatory beta(3)-component which displayed monophasic Michaelis-Menten kinetics. The inhibitory alpha(1)-component (which was also monophasic but had a 2-fold higher EC(50)) was mediated via an increase in [Ca(2+)](i); the protein kinase C pathway was not involved. The [Ca(2+)](i) increase which resulted in massive inhibition of cAMP accumulation was very low: <100 nM. The [Ca(2+)](i) signal stimulated a calmodulin-controlled phosphodiesterase, possibly PDE-1. The acquirement of this specific interaction pattern between beta- and alpha(1)-adrenergic stimulation was thus part of the differentiation program of the brown adipocytes. It was concluded that an array of synergistic or inhibitory alpha(1)/beta interactions occur in the adrenergic regulation of this cell type which is unique in its dependence upon adrenergic stimulation for cellular proliferation, differentiation, and metabolic function.

The bioenergetics of brown fat mitochondria isolated from UCP1-ablated mice were investigated. The mitochondria had lost the high GDP-binding capacity normally found in brown fat mitochondria, and they were innately in an energized state, in contrast to wild-type mitochondria. GDP, which led to energization of wild-type mitochondria, was without effect on the brown fat mitochondria from UCP1-ablated mice. The absence of thermogenic function did not result in reintroduction of high ATP synthase activity. Remarkably and unexpectedly, the mitochondria from UCP1-ablated mice were as sensitive to the de-energizing ("uncoupling") effect of free fatty acids as were UCP1-containing mitochondria. Therefore, the de-energizing effect of free fatty acids does not appear to be mediated via UCP1, and free fatty acids would not seem to be the intracellular physiological activator involved in mediation of the thermogenic signal from the adrenergic receptor to UCP1. In the UCP1-ablated mice, Ucp2 mRNA levels in brown adipose tissue were 14-fold higher and Ucp3 mRNA levels were marginally lower than in wild-type. The Ucp2 and Ucp3 mRNA levels were therefore among the highest found in any tissue. These high mRNA levels did not confer on the isolated mitochondria any properties associated with de-energization. Thus, the mere observation of a high level of Ucp2 or Ucp3 mRNA in a tissue cannot be taken as an indication that mitochondria isolated from that tissue will display innate de-energization or thermogenesis.

Looks at some of country music's top songwriters, including Harlan Howard, Whitey Shafer and Pat Alger. Discusses the local support writers find in Nashville. Collaboration; Difficulties; Broadcasting song focus; Common components. INSET: Fine lines (the best country song titles ever), by M. Hume; Blue.

Brown fat hypertrophy in the rat resulting from cold adaptation is shown here to involve increased mitochondrial, peroxisomal, and lysosomal enzyme activities. Mitochondrial activity in homogenates of brown fat was estimated as cytochrome c oxidase. After 4 wk in the cold (+5 C), the total activity was 3-fold higher than in control rats, although the specific activity was somewhat lower. Peroxisomal activity was followed as cyanide-insensitive palmitoyl-CoA-dependent NAD+ reduction (palmitoyl-CoA oxidase) and as catalase. The total activity of both palmitoyl-CoA oxidase and catalase was more than 10-fold higher than in controls and the specific activity about 3-fold higher. Acid phosphatase, used as a lysosomal marker, showed a 6-fold higher total activity and almost twice as high specific activity. The relatively greater increase in peroxisomes and lysosomes compared with mitochondria indicates an involvement in thermogenesis also for these organelles.

The use of two low-power, single-mode cw dye lasers and a high-power cw Nd^+3:YAG laser is shown to provide high sensitivity and high selectivity as an ionization source for mass spectrometry. The sensitivity for half of the upper states investigated is greater than with electron-impact ionization, and isotopic selectivities of 800 are observed.

Photon-correlation spectroscopy, using both time-interval distribution and burst multiplicity measurements, has been used to investigate the 460.7-nm resonance line of strontium in a low-density atomic beam. A model that considers transit times and thermal velocity distributions for the interval measurements is presented and compared with experimental results. The measurements can determine detection efficiency and absolute beam densities and evaluate nonstatistical behavior such as photomultiplier afterpulsing and dark-count correlation. Photon correlation provides substantial improvements in spectroscopic measurements: linewidths less than the natural lifetime limit are observed, and suppression of Lorentzian tails reduces isotopic interferences by several orders of magnitude. Signal-to-background noise ratios are also improved, with detection limits (1-sec integration) of 10 atoms per second.

We deflected a well-collimated beam of krypton atoms in the metastable ls_5 state by radiation pressure from a single-frequency dye laser. To produce the radiation pressure, we resonantly excited the krypton atoms, using the 1s_5–2p_9 transition. The natural width of this transition is much smaller than its isotope shift, which allowed us to deflect one isotope at a time. This created a new isotopically enriched atomic beam of Kr(1s_5). We achieved a maximum enrichment factor of 1.2 × 10^4 at a deflection angle of 19 mrad. This enrichment is limited mainly by scattering of the parent atomic beam by residual gas. This degree of enrichment may make it possible to perform sensitive measurements of the concentrations of rare krypton isotopes in environmental samples.

We have identified a previously uncharacterized gene that is implicated in the thermogenic function of brown adipose tissue of mice. This gene, termed Cig30, is the first mammalian member of a novel gene family comprising several nematode and yeast genes, such as SUR4 and FEN1, mutation of which is associated with highly pleiotropic phenotypes. It codes for a 30-kDa plasma membrane glycoprotein with five putative transmembrane domains. The Cig30 mRNA was readily detected only in brown fat and liver. When animals were exposed to a 3-day cold stress, the Cig30 expression was selectively elevated in brown fat more than 200-fold. Similar increases were brought about in two other conditions of brown fat recruitment, namely during perinatal development and after cafeteria diet. The magnitude of Cig30 mRNA induction in the cold could be mimicked by chronic norepinephrine treatment in vivo. However, in primary cultures of brown adipocytes, a synergistic action of norepinephrine and dexamethasone was required for full expression of the gene, indicating that both catecholamines and glucocorticoids are required for the induction of Cig30. We propose that the CIG30 protein is involved in a pathway connected with brown fat hyperplasia.

The relationship between cAMP levels and thermogenesis was investigated in brown fat cells from Syrian hamsters. Irrespective of whether the selective beta3-, beta2-, and beta1-agonists BRL 37344, salbutamol, and dobutamine or the physiological agonist norepinephrine was used to stimulate the cells, increases in cAMP levels were mediated via the beta3-receptor, as were the thermogenic effects. However, the relationship "thermogenesis per cAMP" was much lower for agents other than norepinephrine. Similarly, forskolin, although more potent than norepinephrine in elevating cAMP, was less potent in inducing thermogenesis. The selective alpha1-agonist cirazoline was in itself without effect on cAMP levels or thermogenesis, but when added to forskolin-stimulated cells, potentiated thermogenesis, up to the norepinephrine level, without affecting cAMP. This potentiation could not be inhibited by chelerythrine, but could be mimicked by Ca2+ ionophores. It was apparently not mediated via calmodulin-dependent protein kinase and was not an effect on mitochondrial respiratory control. The ability of all cAMP-elevating agents to induce thermogenesis in brown fat cells has earlier been interpreted to mean that it is only through the beta-receptors and the resulting increase in cAMP levels that thermogenesis is induced. However, it is here concluded that the thermogenic response to norepinephrine involves two interacting parts, one mediated via beta-receptors and cAMP and the other via alpha1-receptors and increases in cytosolic Ca2+ levels.

The regulation of the expression of the beta3 adrenoreceptor gene was examined in the brown adipose tissue of intact mice and in murine brown fat primary cell cultures. Both in vivo and in vitro, high levels of beta3 receptor mRNA were observed. Acute cold exposure of mice resulted in a marked and rapid down-regulation of beta3 gene expression; this down-regulation was, however, transient. Similarly, in brown fat cell cultures, norepinephrine addition led to down-regulation of beta3 gene expression, with a lag phase of 30 min and with an apparent half-life of beta3 mRNA of approximately 30 min. This down-regulation was stimulated via the beta3 receptors themselves and mediated via cAMP; the apparent affinity of norepinephrine was extremely high (<1 nM). The degradation rate after actinomycin was identical to that after norepinephrine and was not affected by the presence of norepinephrine; thus, the down-regulation was due to cessation of transcription but not to an increased rate of degradation. Notably, inhibition of protein synthesis by cycloheximide also led to down-regulation. The norepinephrine-induced down-regulation was transient; spontaneous recovery occurred after approximately 18 h and was not due to depletion of adrenergic agent. Recovery did not occur in the presence of cycloheximide. After recovery, the cells showed a functional desensitization of the down-regulation process itself (EC50 now approximately 10 nM). It is concluded that a down-regulated state cannot explain the functional desensitization of beta3 adrenergic responsiveness observed in brown fat cells isolated from cold-acclimated animals (i.e. physiologically chronically adrenergically stimulated brown fat cells); since the beta3 receptor is not subject to desensitization via phosphorylation processes, no satisfactory explanation for the functional desensitization exists as yet. A model is presented for the down-regulation/recovery process, involving the participation of a phosphorylatable short-lived transcription factor.

Brown fat mitochondria were isolated from cold-exposed and control rats, and their content of the brown-fat-specific 32-kDa “uncoupling” protein thermogenin determined both by the traditional [3H]GDP-binding method and by the recently developed enzyme-linked immunosorbent assay (ELISA). In mitochondria isolated from both cold-acclimated (3 wk at 4 degrees C) and cold-exposed rats (24 h), an increase in thermogenin content was observable, both when estimated by the [3H]GDP-binding method and by the ELISA assay, and there was no statistically significant difference in the magnitude of these increases in the two methods. In 1 h cold-exposed rats there was no increase in [3H]GDP binding or in the ELISA reaction. When the amount of thermogenin was plotted against [3H]GDP binding in the different states, a relationship of 75,000 g thermogenin per mole GDP bound was obtained. Based on the resolution of these two methods, and under the three conditions investigated, it was concluded that there was no reason to postulate the existence of a “masked” form of thermogenin or of an “unmasking” process and that thermogenin in the mitochondria, as in the isolated state, has apparently one GDP binding site per dimer.

The ability of adrenergic agents to promote the differentiation and especially the mitochondriogenesis of brown fat precursor cells, grown in culture, was investigated. These cells begin to differentiate during the days preceding confluence. We found here that, already during the early growth phase, the cultures (essentially precursor cells and preadipocytes at this stage) show increased cyclic AMP (cAMP) levels when acutely stimulated with norepinephrine (NE). The cultured cells were therefore chronically treated with NE up to the time of confluence, and their cytochrome-c oxidase activity was measured as an index of mitochondriogenesis. Chronic NE treatment resulted in an increased cytochrome-c oxidase activity of the cells at confluence. This effect was reproduced by selective activation of adenylate cyclase with cholera toxin, suggesting that the NE effect was exerted through an increase in cAMP. Ascorbate (added with NE as an antioxidant) had in itself a positive effect, both on final cell number and on cytochrome-c oxidase activity. It is concluded that NE, working through beta-adrenergic receptors, can stimulate mitochondriogenesis in brown fat cells through a direct effect on the cells, in accordance with the suggestion (based on in vivo experiments) that NE accelerates, rather than initiates, the differentiation process.

Coupled mitochondria of brown adipose tissue can accumulate Ca2+ if a substrate is present. The Ca2+ is released by addition of 20 mM Na+, but not by addition of K+ or choline +. Energy-dissipating Na+-induced Ca2+ cycling occurs maximally with 20 mM Na+ and 10 microM Ca2+. In brown adipocytes, the Ca2+ ionophore A23187 and the Na+ ionophore monensin increase respiration if substrate is added, and incubation in a low-Na+ buffer decreases norepinephrine-induced respiration. Thus Na+-induced Ca2+ release takes place in brown adipose tissue; released Ca2+ could have a regulatory or thermogenic role or both.

In order to investigate the postnatal recruitment process, gene expression in the brown adipose tissue of rat pups was followed during the first 20 h of life. In normal pups, the level of mRNA coding for the uncoupling protein thermogenin increased markedly but gradually within the first 24 h. Lipoprotein lipase and actin mRNA levels were relatively low and remained constant. In pups exposed to thermoneutral temperature (35 degrees C) for the first 12 h after birth, no increase in thermogenin mRNA or lipoprotein lipase mRNA was observed, whereas in pups exposed to 28 degrees C a clear increase in both thermogenin and lipoprotein lipase mRNA levels was found. Actin mRNA levels were not affected by the environmental temperature under these circumstances. It was concluded that the postnatal recruitment in brown adipose tissue is a consequence of the cold stress experienced by the newborn pups. Thus, postnatal recruitment is not ontogenically predetermined.

We have investigated several effects of optical pumping in the hyperfine spectrum of the6s6p1P1?6s21S0 transition in137Ba. Most of these effects are explained by absorption strength changes which occur because of redistribution of population among magnetic substates. At very low laser intensities, no redistribution effects are observed. At higher intensities, it is possible to either empty the magnetic substates that are accessible to optical excitation, or to redistribute the population among these states until a steady-state condition is achieved. The first case results in the familiar disappearance of a hyperfine line. The less familiar second case can result in peak-height ratios in the Ba1P1?1S0 hyperfine spectrum that differ by almost a factor of three from the low-intensity case. In this second case, the observed linewidth can either broaden or narrow, depending on whether redistribution decreases or increases absorption strength. At high intensities, saturation effects dominate and branching to intermediateD states becomes apparent. We report here the result of a numerically integrated rate equation model which shows good agreement with our experiments.

The thermogenic capacity of brown fat from neonatal and developing hamsters was investigated. The method used was to measure the capacity of brown fat mitochondria to bind externally added guanosine diphosphate (GDP). This gives an estimate of the number of proton-conducting channels and hence the capacity of heat production in the mitochondria. At an age of 12 days post-partum the GDP-binding capacity is low: 0.14 nmol GDP/mg mitochondrial protein. Thereafter the capacity shows a steady increase up to 0.54 nmol/mg at 20 days. The peak is followed by a slow decrease down to the level of the adult hamster: 0.32 nmol/mg. This pattern of brown fat development is strikingly similar to reports on the development of oxygen consumption measured on whole animals or on the ability to maintain a constant body temperature when the ambient temperature is lowered. The calorigenic response to injected noradrenaline also follows this pattern. It is therefore justified to suggest that brown fat is a major effector of regulative metabolic heat production in the developing hamster.

A low content of mitochondrial ATPase in brown adipose tissue (BAT) has previously been found to contrast with high levels of the transcripts of the beta-subunit of the F1 part of the ATPase and of the transcripts of the mitochondrial encoded subunits (Houstĕk, J., Tvrdík, P., Pavelka, S., and Baudysová, M. (1991) FEBS Lett. 294, 191-194). To delineate which subunit limits the synthesis of the ATPase complex, we have studied the expression of the nuclear genes encoding subunits alpha, beta, and gamma of the catalytic F1 part and the b, c, d, and OSCP subunits of the F0 part of the ATPase. In comparison with other tissues of mice, high levels of transcripts of alpha-F1, beta-F1, gamma-F1, b-F0, d-Fo, and OSCP were found in BAT. The only genes expressed at a low level in BAT were those of the c-F0 subunit. The levels of c-F0 transcripts were 4-70-fold lower in BAT than in other tissues. An analogous expression pattern of the ATPase genes was found in BAT of adult rat and hamster. In BAT of newborn lamb, which, in contrast to other mammals, has a high content of mitochondrial ATPase, correspondingly high levels of c-F0 mRNA were found Expression of the c-F0 genes also correlated well with the ontogenic development of BAT in the hamster, being high during the first postnatal week when mitochondria are nonthermogenic and contain a relatively high amount of ATPase, but low on subsequent days when ATPase content decreases, as the thermogenic function develops. It is suggested that expression of the c-F0 genes and subsequent synthesis of the hydrophobic subunit c of the membrane-intrinsic F0 part of the enzyme may control the biosynthesis of the ATPase complex in BAT. An analogous regulatory role of the c-F0 subunit could be postulated in other tissues.

To investigate the cellular control of the recruitment process in brown adipose tissue, the ability of cholera toxin to influence the differentiation of brown preadipocytes developing in culture was investigated. Stromalvascular cells obtained from the brown adipose tissue of 3-wk-old rats were grown in culture for 6-7 days in the presence or absence of cholera toxin. It was found that cholera toxin treatment decreased the expression of the actin gene (indicating an increased degree of differentiation), while at the same time promoting the expression of the genes coding for the mitochondriogenesis marker cytochrome-c oxidase and for the adipocyte conversion marker lipoprotein lipase (all followed at the mRNA level). Chronic cholera toxin treatment also increased the total amount of protein per cell in culture, and a specific cholera toxin-induced 35-kDa protein was identified. It was concluded that (in contrast to the case suggested for white preadipocytes) cholera toxin treatment of brown preadipocytes may not only affect the activity of catabolic enzymes but may also directly promote the differentiation process, indicating that this process is under beta-adrenergic control in the adapting animal.

AbstractSchultenite, PbHAsO4, known only from Tsumeb, Namibia, has been identified from a second occurrence: near North Bend, King County, Washington, USA. It occurs as euhedral crystals in a quartz-arsenopyrite-galena vein. It has a measured density of 6.07(3) g/cm3and a calculated density of 6.079(4) g/cm3. The white to colourless crystals have a white streak, adamantine lustre, and fluoresce dull yellow under long wave ultraviolet light. Schultenite is monoclinic. Paor P2/a; the unit cell parameters refined from the X-ray powder diffraction data are: a5.827(3), b6.743(3), c4.847(3) Å, ß95.34(5)°; V 189.6(1) Å3; Z = 2; a:b:c= 0.8642:1:0.7188. The forms {010}, {001}, and {3¯22} were observed on the Washington schultenite crystals.