A Simplified Mass-Transfer Model for Visual Pigments in Amphibian Retinal-Cone Outer Segments

When radiolabeled precursors and autoradiography are used to investigate turnover of protein components in photoreceptive cone outer segments (COSs), the labeled components—primarily visual pigment molecules (opsins)—are diffusely distributed along the COS. To further assess this COS labeling pattern, we derive a simplified mass-transfer model for quantifying the contributions of advective and diffusive mechanisms to the distribution of opsins within COSs of the frog retina. Two opsin-containing regions of the COS are evaluated: the core axial array of disks and the plasmalemma. Numerical solutions of the mass-transfer model indicate three distinct stages of system evolution. In the first stage, plasmalemma diffusion is dominant. In the second stage, the plasmalemma density reaches a metastable state and transfer between the plasmalemma and disk region occurs, which is followed by an increase in density that is qualitatively similar for both regions. The final stage consists of both regions slowly evolving to the steady-state solution. Our results indicate that autoradiographic and cognate approaches for tracking labeled opsins in the COS cannot be effective methodologies for assessing new disk formation at the base of the COS.     

Three-way Interaction between 14-3-3 Proteins, the N-terminal Region of Tyrosine Hydroxylase, and Negatively Charged Membranes

Tyrosine hydroxylase (TH), the rate-limiting enzyme in the synthesis of catecholamines, is activated by phosphorylation-dependent binding to 14-3-3 proteins. The N-terminal domain of TH is also involved in interaction with lipid membranes. We investigated the binding of the N-terminal domain to its different partners, both in the unphosphorylated (TH-(1–43)) and Ser¹⁹-phosphorylated (THp-(1–43)) states by surface plasmon resonance. THp-(1–43) showed high affinity for 14-3-3 proteins (K_d ∼ 0.5 μm for 14-3-3γ and -ζ and 7 μm for 14-3-3η). The domains also bind to negatively charged membranes with intermediate affinity (concentration at half-maximal binding S_0.5 = 25–58 μm (TH-(1–43)) and S_0.5 = 135–475 μm (THp-(1–43)), depending on phospholipid composition) and concomitant formation of helical structure. 14-3-3γ showed a preferential binding to membranes, compared with 14-3-3ζ, both in chromaffin granules and with liposomes at neutral pH. The affinity of 14-3-3γ for negatively charged membranes (S_0.5 = 1–9 μm) is much higher than the affinity of TH for the same membranes, compatible with the formation of a ternary complex between Ser¹⁹-phosphorylated TH, 14-3-3γ, and membranes. Our results shed light on interaction mechanisms that might be relevant for the modulation of the distribution of TH in the cytoplasm and membrane fractions and regulation of l-DOPA and dopamine synthesis.     

Distal Histidine Stabilizes Bound O2 and Acts as a Gate for Ligand Entry in Both Subunits of Adult Human Hemoglobin

The role of the distal histidine in regulating ligand binding to adult human hemoglobin (HbA) was re-examined systematically by preparing His(E7) to Gly, Ala, Leu, Gln, Phe, and Trp mutants of both Hb subunits. Rate constants for O₂, CO, and NO binding were measured using rapid mixing and laser photolysis experiments designed to minimize autoxidation of the unstable apolar E7 mutants. Replacing His(E7) with Gly, Ala, Leu, or Phe causes 20–500-fold increases in the rates of O₂ dissociation from either Hb subunit, demonstrating unambiguously that the native His(E7) imidazole side chain forms a strong hydrogen bond with bound O₂ in both the α and β chains (ΔG_{His(E7)H-bond} ≈ −8 kJ/mol). As the size of the E7 amino acid is increased from Gly to Phe, decreases in k_O2′, k_NO′, and calculated bimolecular rates of CO entry (k_entry′) are observed. Replacing His(E7) with Trp causes further decreases in k_O2′, k_NO′, and k_entry′ to 1–2 μm⁻¹ s⁻¹ in β subunits, whereas ligand rebinding to αTrp(E7) subunits after photolysis is markedly biphasic, with fast k_O2′, k_CO′, and k_NO′ values ≈150 μm⁻¹ s⁻¹ and slow rate constants ≈0.1 to 1 μm⁻¹ s⁻¹. Rapid bimolecular rebinding to an open α subunit conformation occurs immediately after photolysis of the αTrp(E7) mutant at high ligand concentrations. However, at equilibrium the closed αTrp(E7) side chain inhibits the rate of ligand binding >200-fold. These data suggest strongly that the E7 side chain functions as a gate for ligand entry in both HbA subunits.     

Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Content, Assimilatory Charge, and Mesophyll Conductance in Leaves

The content of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) (E_t; EC 4.1.1.39) measured in different-aged leaves of sunflower (Helianthus annuus) and other plants grown under different light intensities, varied from 2 to 75 μmol active sites m⁻². Mesophyll conductance (μ) was measured under 1.5% O₂, as well as postillumination CO₂ uptake (assimilatory charge, a gas-exchange measure of the ribulose-1,5-bisphosphate pool). The dependence of μ on E_t saturated at E_t = 30 μmol active sites m⁻² and μ = 11 mm s⁻¹ in high-light-grown leaves. In low-light-grown leaves the dependence tended toward saturation at similar E_t but reached a μ of only 6 to 8 mm s⁻¹. μ was proportional to the assimilatory charge, with the proportionality constant (specific carboxylation efficiency) between 0.04 and 0.075 μm⁻¹ s⁻¹. Our data show that the saturation of the relationship between E_t and μ is caused by three limiting components: (a) the physical diffusion resistance (a minor limitation), (b) less than full activation of Rubisco (related to Rubisco activase and the slower diffusibility of Rubisco at high protein concentrations in the stroma), and (c) chloroplast metabolites, especially 3-phosphoglyceric acid and free inorganic phosphate, which control the reaction kinetics of ribulose-1,5-bisphosphate carboxylation by competitive binding to active sites.Rubisco (EC 4.1.1.39) catalyzes the irreversible carboxylation of RuBP to form two PGA molecules (in this work the oxygenase reaction was not active since a low O₂ concentration was used). RuBP carboxylation is the major rate-determining reaction in photosynthetic CO₂ assimilation. All factors that influence the photosynthetic rate do so by influencing the activity of Rubisco and the concentration of its substrates, CO₂ and RuBP. E_t in leaves may be as high as 75 μmol m⁻², and for the extracted enzyme K_m(CO₂) = 9.4 μm (Makino et al., 1985a) and K_m(RuBP) = 30 to 40 μm (Yeoh et al., 1981). In leaves photosynthesizing under atmospheric conditions, the concentration of RuBP may increase to 10 to 15 mm (Badger et al., 1984; Sharkey et al., 1986), but the concentration of CO₂ is usually about 4 to 8 μm in leaf intercellular spaces, depending on stomatal conductance. This CO₂ concentration is well below the K_m(CO₂) of the enzyme, and it is the initial slope of the kinetic curve V_M/K_m(CO₂), termed carboxylation conductance, that becomes important.r_c limits the CO₂-fixation rate in series with the other resistances, r_g and r_md. The carboxylation rates are usually expressed in relation to C_i or C_w. C_c is usually about 20% to 30% lower than C_w because of concentration decrease generated by the carboxylation flux on r_md. Considering the above, the carboxylation conductance in intact leaves in vivo may be found as the initial slope of the A versus C_c graph at low C_c values. If C_c cannot be calculated because r_md is unknown, the closest approximation is a plot of A versus C_w or A versus C_i. The true parameters of the carboxylase can be found only from experiments carried out in nonphotorespiratory conditions (1%–2% O₂); otherwise the competing oxygenase reaction consumes a part of RuBP and partially inhibits carboxylase activity.Because of technical problems with the measurement of A versus C_w relationships, in many studies only the net photosynthetic rate under atmospheric conditions (21% O₂) was related to Rubisco activity or content. Nevertheless, good correlation has been found (Makino et al., 1983; Hudson et al., 1992; Jacob and Lawlor, 1992; Jiang and Rodermel, 1995; Nakano et al., 1997). These results indicated that the level of Rubisco protein could be a limiting factor in photosynthesis throughout the life span of the leaf under natural environmental conditions. On the other hand, when Rubisco levels in leaves exceeded 4 g m⁻² (60 μmol m⁻²), the in vivo Rubisco activity (measured as photosynthesis under pC_i = 20 to 30 Pa and 21% O₂) became curvilinearly correlated with E_t (Makino et al., 1994, 1997). When measurements were made over the whole life span of wheat leaves, the measured rates of photosynthesis were lower in young leaves, which had high protein content, than would have been expected from the amount and activity of Rubisco (Lawlor et al., 1989).During senescence the decrease in Rubisco activity was initially greater than the decrease in net photosynthesis (Hall et al., 1978). In a willow canopy, Rubisco-specific activity was higher when the apparent E_t (N content in leaves) was smaller (Vapaavuori and Vuorinen, 1989). A similar nonlinearity was found in our previous experiments (Eichelmann and Laisk, 1990), in which we obtained a saturating relationship when E_t exceeded 30 μmol m⁻². In the latter work the initial slope of the A versus C_w curves under nonphotorespiratory conditions (1.5% O₂) was assumed to represent the Rubisco activity in vivo and was compared with the E_t. We discovered that growth light had the strongest influence on the saturation of the relationship between μ and E_t. In the present work we present insight into this relationship, using not only plants grown under different light intensities but also leaves adapted to different light intensities.     

Description of Hemicaloosia graminis n. sp. (Nematoda: Caloosiidae) Associated with Turfgrasses in North and South Carolina,USA

A new nematode species was discovered during a diversity survey of plant-parasitic nematodes on turfgrass conducted in North and South Carolina in 2010 and 2011. It is described herein as Hemicaloosia graminis n. sp. and is characterized by two annuli in the lip region, one lateral line, body 610.0–805.0 μm long, stylet 65.0–74.6 μm long, vulva at 84.1% –85.8% of the body , 254–283 annuli, vulva at the 38–53^rd annulus from tail terminus, 12–14 annuli between vulva and anus, tail elongate-pointed, 67.5–84.8 μm long in females and spicule straight, 31.0 μm long, caudal alae well developed, two lateral lines in males. The newly described species is morphologically closest to H. paradoxa, but has a longer stylet (65.0–74.6 vs 61.0–65.0 μm) and a higher V-value (84.1–85.8 vs 78.1–84.0%), less RV (38–53 vs 50–56), higher RVan (12–14 vs 10) in females, and a shorter tail (30.1 vs 36.7 μm) and more anteriorly located excretory pore (105.9 vs 140.0 μm) in the male. It was easily differentiated from other species based on near-full-length small subunit rRNA gene (SSU) and ITS1 sequences. Phylogenetic analysis from SSU supports placement in a monophyletic clade with the genus Caloosia. An identification key and a table of distinguishing characteristics are presented for all seven species of Hemicaloosia.     

Sucrose Octasulfate Selectively Accelerates Thrombin Inactivation by Heparin Cofactor II

Inactivation of thrombin (T) by the serpins heparin cofactor II (HCII) and antithrombin (AT) is accelerated by a heparin template between the serpin and thrombin exosite II. Unlike AT, HCII also uses an allosteric interaction of its NH₂-terminal segment with exosite I. Sucrose octasulfate (SOS) accelerated thrombin inactivation by HCII but not AT by 2000-fold. SOS bound to two sites on thrombin, with dissociation constants (K_D) of 10 ± 4 μm and 400 ± 300 μm that were not kinetically resolvable, as evidenced by single hyperbolic SOS concentration dependences of the inactivation rate (k_obs). SOS bound HCII with K_D 1.45 ± 0.30 mm, and this binding was tightened in the T·SOS·HCII complex, characterized by K_complex of ∼0.20 μm. Inactivation data were incompatible with a model solely depending on HCII·SOS but fit an equilibrium linkage model employing T·SOS binding in the pathway to higher order complex formation. Hirudin-(54–65)(SO₃⁻) caused a hyperbolic decrease of the inactivation rates, suggesting partial competitive binding of hirudin-(54–65)(SO₃⁻) and HCII to exosite I. Meizothrombin(des-fragment 1), binding SOS with K_D = 1600 ± 300 μm, and thrombin were inactivated at comparable rates, and an exosite II aptamer had no effect on the inactivation, suggesting limited exosite II involvement. SOS accelerated inactivation of meizothrombin 1000-fold, reflecting the contribution of direct exosite I interaction with HCII. Thrombin generation in plasma was suppressed by SOS, both in HCII-dependent and -independent processes. The ex vivo HCII-dependent process may utilize the proposed model and suggests a potential for oversulfated disaccharides in controlling HCII-regulated thrombin generation.     

Cloning, Expression in Escherichia coli, and Characterization of Arabidopsis thaliana UMP/CMP Kinase

A cDNA encoding the Arabidopsis thaliana uridine 5′-monophosphate (UMP)/cytidine 5′-monophosphate (CMP) kinase was isolated by complementation of a Saccharomyces cerevisiae ura6 mutant. The deduced amino acid sequence of the plant UMP/CMP kinase has 50% identity with other eukaryotic UMP/CMP kinase proteins. The cDNA was subcloned into pGEX-4T-3 and expressed as a glutathione S-transferase fusion protein in Escherichia coli. Following proteolytic digestion, the plant UMP/CMP kinase was purified and analyzed for its structural and kinetic properties. The mass, N-terminal sequence, and total amino acid composition agreed with the sequence and composition predicted from the cDNA sequence. Kinetic analysis revealed that the UMP/CMP kinase preferentially uses ATP (Michaelis constant [K_m] = 29 μm when UMP is the other substrate and K_m = 292 μm when CMP is the other substrate) as a phosphate donor. However, both UMP (K_m = 153 μm) and CMP (K_m = 266 μm) were equally acceptable as the phosphate acceptor. The optimal pH for the enzyme is 6.5. P¹, P⁵-di(adenosine-5′) pentaphosphate was found to be a competitive inhibitor of both ATP and UMP.     

Characterization of Aminopeptidase in the Free-living Nematode Panagrellus redivivus: Subcellular Distribution and Possible Role in Neuropeptide Metabolism

Aminopeptidase was detected in homogenates of the free-living nematode Panagrellus redivivus with the aminoacyl substrate L-alanine-4-nitroanilide. Subcellular distribution of activity was 80% soluble and 20% membrane-associated. Aminopeptidases in the two fractions differed in affinity for Ala-4-NA, with Km''s of 0.65 mM (soluble) and 2.90 mM (membrane). Specific activities (units/mg) at pH 7.8, 27°C were 9.10 (soluble) and 14.30 (membrane). Each enzyme was competitively inhibited by amastatin (90% at 100 μM inhibitor, IC₅₀ = 3.7 μM) and inhibited by puromycin (30% at 500 μM) and 1,10-phenanthroline (IC_50''s:; 148 μM, soluble; 89 μM, membrane). Activity was restored by Zn⁺⁺, with maximum recoveries of 50% (soluble) and 90% (membrane), each at 23 μM ZnCl₂. Estimated molecular masses for each were ∼150 kDa. FMRFamide-like neuropeptides behaved as competitive inhibitors. Modification of the N-terminal F of FMRFamide weakened inhibition by 95%, suggesting that the N-terminus is essential for binding to the enzyme. Two nematode FMRFamides, APKPFIRFa and RNKFEFIRFa, were the most potent tested. This is the first biochemical characterization of aminopeptidase in a free-living nematode other than Caenorhabditis elegans and demonstrates the high selectivity of the P. redivivus enzymes for neuropeptide substrates.     

Sex-dependent influences of obesity on cerebral white matter investigated by diffusion-tensor imaging

Several studies have shown that obesity is associated with changes in human brain function and structure. Since women are more susceptible to obesity than men, it seems plausible that neural correlates may also be different. However, this has not been demonstrated so far. To address this issue, we systematically investigated the brain''s white matter (WM) structure in 23 lean to obese women (mean age 25.5 y, std 5.1 y; mean body mass index (BMI) 29.5 kg/m², std 7.3 kg/m²) and 26 lean to obese men (mean age 27.1 y, std 5.0 y; mean BMI 28.8 kg/m², std 6.8 kg/m²) with diffusion-weighted magnetic resonance imaging (MRI). There was no significant age (p>0.2) or BMI (p>0.7) difference between female and male participants. Using tract-based spatial statistics, we correlated several diffusion parameters including the apparent diffusion coefficient, fractional anisotropy (FA), as well as axial (λ_∥) and radial diffusivity (λ_⊥) with BMI and serum leptin levels. In female and male subjects, the putative axon marker λ_∥ was consistently reduced throughout the corpus callosum, particularly in the splenium (r = −0.62, p<0.005). This suggests that obesity may be associated with axonal degeneration. Only in women, the putative myelin marker λ_⊥ significantly increased with increasing BMI (r = 0.57, p<0.005) and serum leptin levels (r = 0.62, p<0.005) predominantly in the genu of the corpus callosum, suggesting additional myelin degeneration. Comparable structural changes were reported for the aging brain, which may point to accelerated aging of WM structure in obese subjects. In conclusion, we demonstrate structural WM changes related to an elevated body weight, but with differences between men and women. Future studies on obesity-related functional and structural brain changes should therefore account for sex-related differences.     

Protein 4.2 Binds to the Carboxyl-terminal EF-hands of Erythroid ��-Spectrin in a Calcium- and Calmodulin-dependent Manner

Spectrin and protein 4.1 cross-link F-actin protofilaments into a network called the membrane skeleton. Actin and 4.1 bind to one end of β-spectrin. The adjacent end of α-spectrin, called the EF-domain, is calmodulin-like, with calcium-dependent and calcium-independent EF-hands. It has no known function. However, the sph^1J/sph^1J mouse has very fragile red cells and lacks the last 13 amino acids in the EF-domain, suggesting the domain is critical for skeletal integrity. Using pulldown binding assays, we find the α-spectrin EF-domain either alone or incorporated into a mini-spectrin binds native and recombinant protein 4.2 at a previously identified region of 4.2 (G₃ peptide). Native 4.2 binds with an affinity comparable with other membrane skeletal interactions (K_d = 0.30 μm). EF-domains bearing the sph^1J mutation are inactive. Binding of protein 4.2 to band 3 (K_d = 0.45 μm) does not interfere with the spectrin-4.2 interaction. Spectrin-4.2 binding is amplified by micromolar concentrations of Ca²⁺ (but not Mg²⁺) by three to five times. Calmodulin also binds to the EF-domain (K_d = 17 μm), and Ca²⁺-calmodulin blocks Ca²⁺-dependent binding of protein 4.2 but not Ca²⁺-independent binding. The data suggest that protein 4.2 is located near protein 4.1 at the spectrin-actin junctions. Because proteins 4.1 and 4.2 also bind to band 3, the erythrocyte anion channel, we suggest that one or both of these proteins cause a portion of band 3 to localize near the spectrin-actin junctions and provide another point of attachment between the membrane skeleton and the lipid bilayer.     

Successional Development of Sulfate-Reducing Bacterial Populations and Their Activities in a Wastewater Biofilm Growing under Microaerophilic Conditions

A combination of fluorescence in situ hybridization, microprofiles, denaturing gradient gel electrophoresis of PCR-amplified 16S ribosomal DNA fragments, and 16S rRNA gene cloning analysis was applied to investigate successional development of sulfate-reducing bacteria (SRB) community structure and in situ sulfide production activity within a biofilm growing under microaerophilic conditions (dissolved oxygen concentration in the bulk liquid was in the range of 0 to 100 μM) and in the presence of nitrate. Microelectrode measurements showed that oxygen penetrated 200 μm from the surface during all stages of biofilm development. The first sulfide production of 0.32 μmol of H₂S m⁻² s⁻¹ was detected below ca. 500 μm in the 3rd week and then gradually increased to 0.70 μmol H₂S m⁻² s⁻¹ in the 8th week. The most active sulfide production zone moved upward to the oxic-anoxic interface and intensified with time. This result coincided with an increase in SRB populations in the surface layer of the biofilm. The numbers of the probe SRB385- and 660-hybridized SRB populations significantly increased to 7.9 × 10⁹ cells cm⁻³ and 3.6 × 10⁹ cells cm⁻³, respectively, in the surface 400 μm during an 8-week cultivation, while those populations were relatively unchanged in the deeper part of the biofilm, probably due to substrate transport limitation. Based on 16S rRNA gene cloning analysis data, clone sequences that related to Desulfomicrobium hypogeium (99% sequence similarity) and Desulfobulbus elongatus (95% sequence similarity) were most frequently found. Different molecular analyses confirmed that Desulfobulbus, Desulfovibrio, and Desulfomicrobium were found to be the numerically important members of SRB in this wastewater biofilm.     

Kinetic Parameters of Denitrification in a River Continuum

Kinetic parameters for nitrate reduction in intact sediment cores were investigated by using the acetylene blockage method at five sites along the Swale-Ouse river system in northeastern England, including a highly polluted tributary, R. Wiske. The denitrification rate in sediment containing added nitrate exhibited a Michaelis-Menten-type curve. The concentration of nitrate for half-maximal activity (K_map) by denitrifying bacteria increased on passing downstream from 13.1 to 90.4 μM in the main river, but it was highest (640 μM) in the Wiske. The apparent maximal rate (V_maxap) ranged between 35.8 and 324 μmol of N m⁻² h⁻¹ in the Swale-Ouse (increasing upstream to downstream), but it was highest in the Wiske (1,194 μmol N m⁻² h⁻¹). A study of nitrous oxide (N₂O) production at the same time showed that rates ranged from below the detection limit (0.05 μmol of N₂O-N m⁻² h⁻¹) at the headwater site to 27 μmol of N₂O-N m⁻² h⁻¹ at the downstream site. In the Wiske the rate was up to 570 μmol of N₂O-N m⁻² h⁻¹, accounting for up to 80% of total N gas production.     

Niosomal Gel of Lornoxicam for Topical Delivery: In vitro Assessment and Pharmacodynamic Activity

Lornoxicam is a potent oxicam class of non steroidal anti-inflammatory agent, prescribed for mild to moderate pain and inflammation. Niosomal gel of lornoxicam was developed for topical application. Lornoxicam niosomes (Lor-Nio) were fabricated by thin film hydration technique. Bilayer composition of niosomal vesicles was optimized. Lor-Nio dispersion was characterized by DSC, XRD, and FT-IR. Morphological evaluation was performed by scanning electron microscopy (SEM). Lor-Nio dispersion was incorporated into a gel using 2% w/w Carbopol 980 NF. Rheological and texture properties of Lor-Nio gel formulation showed suitability of the gel for topical application. The developed formulation was evaluated for in vitro skin permeation and skin deposition studies, occlusivity test and skin irritation studies. Pharmacodynamic activity of the Lor-Nio gel was performed by carragenan-induced rat paw model. Optimized Lor-Nio comprised of Span 60 and cholesterol in a molar ratio of 3:1 with 30 μM dicetyl palmitate as a stabilizer. It had particle size of 1.125 ± 0.212 μm (d₉₀), with entrapment efficiency of 52.38 ± 2.1%. DSC, XRD, and IR studies showed inclusion of Lor into niosomal vesicles. SEM studies showed spherical closed vesicular structure with particles in nanometer range. The in vitro skin permeation studies showed significant improvement in skin permeation and skin deposition for Lor-Nio gel (31.41 ± 2.24 μg/cm², 30.079 ± 1.2 μg/cm²) over plain lornoxicam gel (7.37 ± 1.27 μg/cm², 6.6 ± 2.52 μg/cm²). The Lor-Nio gel formulation showed enhanced anti-inflammatory activity by exhibiting mean edema inhibition (87.69 ± 1.43%) which was significantly more than the plain lornoxicam gel (53.84 ± 2.21%).KEY WORDS: anti-inflammatory activity, lornoxicam, niosomes, rheology, texture analysis     

Imaging atherosclerotic plaque composition with intracoronary optical coherence tomography

Optical coherence tomography (OCT) allows highly accurate diagnosis of atherosclerotic plaques, including measurement of the thickness of fibrous caps, permitting an assessment of the risk of rupture. While the OCT image presents morphological information in highly resolved detail, it relies on interpretation by trained readers for the identification of tissue type. We developed a method for quantitative classification of atherosclerotic plaque constituents. The optical attenuation coefficient μ_t distinguishes different tissue types: necrotic core and macrophage infiltration exhibit strong attenuation, μ_t≥10 mm⁻¹, while calcific and fibrous tissue have a lower μ_t≈2–5 mm⁻¹. (Neth Heart J 2009;17:448-50.)     

Rate Equations and Kinetic Parameters of the Reactions Involved in Pyrite Oxidation by Thiobacillus ferrooxidans

Rate equations and kinetic parameters were obtained for various reactions involved in the bacterial oxidation of pyrite. The rate constants were 3.5 μM Fe²⁺ per min per FeS₂ percent pulp density for the spontaneous pyrite dissolution, 10 μM Fe²⁺ per min per mM Fe³⁺ for the indirect leaching with Fe³⁺, 90 μM O₂ per min per mg of wet cells per ml for the Thiobacillus ferrooxidans oxidation of washed pyrite, and 250 μM O₂ per min per mg of wet cells per ml for the T. ferrooxidans oxidation of unwashed pyrite. The K_m values for pyrite concentration were similar and were 1.9, 2.5, and 2.75% pulp density for indirect leaching, washed pyrite oxidation by T. ferrooxidans, and unwashed pyrite oxidation by T. ferrooxidans, respectively. The last reaction was competitively inhibited by increasing concentrations of cells, with a K_i value of 0.13 mg of wet cells per ml. T. ferrooxidans cells also increased the rate of Fe²⁺ production from Fe³⁺ plus pyrite.     

Mitochondrial Free Ca2+ Levels and Their Effects on Energy Metabolism in Drosophila Motor Nerve Terminals

Mitochondrial Ca²⁺ uptake exerts dual effects on mitochondria. Ca²⁺ accumulation in the mitochondrial matrix dissipates membrane potential (_ΔΨ_m), but Ca²⁺ binding of the intramitochondrial enzymes accelerates oxidative phosphorylation, leading to mitochondrial hyperpolarization. The levels of matrix free Ca²⁺ ([Ca²⁺]_m) that trigger these metabolic responses in mitochondria in nerve terminals have not been determined. Here, we estimated [Ca²⁺]_m in motor neuron terminals of Drosophila larvae using two methods: the relative responses of two chemical Ca²⁺ indicators with a 20-fold difference in Ca²⁺ affinity (rhod-FF and rhod-5N), and the response of a low-affinity, genetically encoded ratiometric Ca²⁺ indicator (D4cpv) calibrated against known Ca²⁺ levels. Matrix pH (pH_m) and _ΔΨ_m were monitored using ratiometric pericam and tetramethylrhodamine ethyl ester probe, respectively, to determine when mitochondrial energy metabolism was elevated. At rest, [Ca²⁺]_m was 0.22 ± 0.04 μM, but it rose to ∼26 μM (24.3 ± 3.4 μM with rhod-FF/rhod-5N and 27.0 ± 2.6 μM with D4cpv) when the axon fired close to its endogenous frequency for only 2 s. This elevation in [Ca²⁺]_m coincided with a rapid elevation in pH_m and was followed by an after-stimulus _ΔΨ_m hyperpolarization. However, pH_m decreased and no _ΔΨ_m hyperpolarization was observed in response to lower levels of [Ca²⁺]_m, up to 13.1 μM. These data indicate that surprisingly high levels of [Ca²⁺]_m are required to stimulate presynaptic mitochondrial energy metabolism.     

Label-free multimodal quantitative imaging flow assay for intrathrombus formation in vitro

Microfluidics in vitro assays recapitulate a blood vessel microenvironment using surface-immobilized agonists under biofluidic flows. However, these assays do not quantify intrathrombus mass and activities of adhesive platelets at the agonist margin and use fluorescence labeling, therefore limiting clinical translation potential. Here, we describe a label-free multimodal quantitative imaging flow assay that combines rotating optical coherent scattering microscopy and quantitative phase microscopy. The combined imaging platform enables real-time evaluation of membrane fluctuations of adhesive-only platelets and total intrathrombus mass under physiological flow rates in vitro. We call this multimodal quantitative imaging flow assay coherent optical scattering and phase interferometry (COSI). COSI records intrathrombus mass to picogram accuracy and shape changes to a platelet membrane with high spatial-temporal resolution (0.4 μm/s) under physiological and pathophysiological fluid shear stress (1800 and 7500 s⁻¹). With COSI, we generate an axial slice of 4 μm from the coverslip surface, approximately equivalent to the thickness of a single platelet, which permits nanoscale quantification of membrane fluctuation (activity) of adhesive platelets during initial adhesion, spreading, and recruitment into a developing thrombus (mass). Under fluid shear, pretreatment with a broad range metalloproteinase inhibitor (250 μM GM6001) blocked shedding of platelet adhesion receptors that shown elevated adhesive platelet activity at average of 42.1 μm/s and minimal change in intrathrombus mass.