Angles of nucleotides bound to cross-bridges in glycerinated muscle fiber at various concentrations of ϵ-ATP, ϵ-ADP and ϵ-AMPPNP detected by polarized fluorescence

The polarized fluorescence from nucleotides bound to myosin heads in glycerinated muscle fibers of rabbit psoas was measured as the number of myosin heads with bound nucleotides was varied by adding various concentrations of fluorescent ?-ATP, ?-ADP and ?-AMPPNP (1:N⁶-etheno-ATP, -ADP and -imido ATP). The angles of the absorption and emission dipoles of bound nucleotides to the fiber axis and their angular distribution were determined from the observed values of four components of the polarized fluorescence.The maximum amount of nucleotides bound to the myosin heads in the fiber, B_m, was 170 to 270 μm. The dissociation constant of nucleotides, $\text{K}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$, increased in the order ?-ATP, ?-ADP, ?-AMPPNP, and was four to six times larger at a sarcomere length (SL) of 2.1 μm than at 3.7 μm.The polarized fluorescence from bound ?-ADP at SL = 2.1 μm was independent of the amount of bound ?-ADP when it was lower than one-half of B_m, indicating a single helical array of myosin heads having ?-ADP. The angles of the absorption dipole, φ_A, and the emission dipole, φ_E, to the fiber axis were 69 ° and 66 °, respectively. As the amount of bound ?-ADP exceeded one-half of B_m, the values of the polarized fluorescence showed that the extra ?-ADP bound to myosin heads with a lower affinity and had different angles to the fiber axis: φ_A and φ_E were 49 ° and 54 °, respectively. The half-maximum width of the angular distribution of these bound ?-ADP molecules, $\text{θ}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$, was about 20 °.During development of isometric tension in the presence of ?-ATP with Mg²⁺, the polarized fluorescence was independent of the amount of bound ?-ATP when it was lower than one-third of B_m or when the concentration of free ?-ATP was lower than 100 μm, indicating a single helical array of myosin heads undergoing the ATPase reaction. The angles of bound nucleotides, φ_A and φ_E, were 68 ° and 64 °, respectively. The half-maximum width of the angular distribution, $\text{θ}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$, was about 22 °. As the amount of bound nucleotides exceeded one-third of B_m, the polarized fluorescence showed deviation from the values expected for the single helical array.The angles φ_A and φ_E for bound ?-AMPPNP were about 58 ° and 62 °, respectively, but the angular distribution was broad; that is, $\text{θ}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ was about 42 °. These angles were independent of the amount of bound ?-AMPPNP.In a stretched fiber with SL = 3.7 μm, the polarized fluorescence showed that the angles of ?-ADP, ?-ATP and ?-AMPPNP bound to myosin heads had almost random distributions; $\text{θ}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ was 90 ° to 100 °, independent of the amount of bound nucleotides. Similar results were obtained with the relaxed fiber in the presence of ?-ATP.     

Spectral microscopic imaging of heterocysts and vegetative cells in two filamentous cyanobacteria based on spontaneous Raman scattering and photoluminescence by 976 nm excitation

Photosynthetic pigment-protein complexes are highly concentrated in thylakoid membranes of chloroplasts and cyanobacteria that emit strong autofluorescence (mainly 600–800?nm). In Raman scattering microscopy that enables imaging of pigment concentrations of thylakoid membranes, near infrared laser excitation at 1064?nm or visible laser excitation at 488–532?nm has been often employed in order to avoid the autofluorescence. Here we explored a new approach to Raman imaging of thylakoid membranes by using excitation wavelength of 976?nm. Two types of differentiated cells, heterocysts and vegetative cells, in two diazotrophic filamentous cyanobacteria, Anabaena variabilis, and Rivularia M-261, were characterized. Relative Raman scattering intensities of phycobilisomes of the heterocyst in comparison with the nearest vegetative cells of Rivularia remained at a significantly higher level than those of A. variabilis. It was also found that the 976?nm excitation induces photoluminescence around 1017–1175?nm from the two cyanobacteria, green alga (Parachlorella kessleri) and plant (Arabidopsis thaliana). We propose that this photoluminescence can be used as an index of concentration of chlorophyll a that has relatively small Raman scattering cross-sections. The Rivularia heterocysts that we analyzed were clearly classified into at least two subgroups based on the Chla-associated photoluminescence and carotenoid Raman bands, indicating two physiologically distinct states in the development or aging of the terminal heterocyst.     

Characterization of the covalent binding of allyl isothiocyanate to β-lactoglobulin by fluorescence quenching,equilibrium measurement,and mass spectrometry

Reversible binding of small compounds through hydrophobic interactions or hydrogen bonding to food proteins (e.g. milk proteins) is a thoroughly researched topic. In contrast, covalent interactions are not well characterized. Here, we report a rare form of positive-cooperativity-linear binding of allyl isothiocyanate with β-lactoglobulin, resulting in the cleavage of a disulfide bond of the protein. We compared three methods (i.e. fluorescence quenching, equilibrium dialysis, and headspace–water equilibrium) to characterize the binding kinetics and investigated the molecular binding by mass spectrometry. The methodologies used were found to be comparable and reproducible in the presence of high and low ligand concentrations for fluorescence quenching and equilibrium-based methods respectively.     

Integrin αMβ2 is differently expressed by subsets of human osteoclast precursors and mediates adhesion of classical monocytes to bone

Bone-degrading osteoclasts are formed through fusion of their monocytic precursors. In the population of human peripheral blood monocytes, three distinct subsets have been identified: classical, intermediate and non-classical monocytes. We have previously shown that when the monocyte subsets are cultured on bone, significantly more osteoclasts are formed from classical monocytes than from intermediate or non-classical monocytes. Considering that this difference does not exist when monocyte subsets are cultured on plastic, we hypothesized that classical monocytes adhere better to the bone surface compared to intermediate and non-classical monocytes. To investigate this, the different monocyte subsets were isolated from human peripheral blood and cultured on slices of human bone in the presence of the cytokine M-CSF. We found that classical monocytes adhere better to bone due to a higher expression of the integrin αMβ2 and that their ability to attach to bone is significantly decreased when the integrin is blocked. This suggests that integrin αMβ2 mediates attachment of osteoclast precursors to bone and thereby enables the formation of osteoclasts.     

Myosin 1b promotes the formation of post-Golgi carriers by regulating actin assembly and membrane remodelling at the trans-Golgi network

The function of organelles is intimately associated with rapid changes in membrane shape. By exerting force on membranes, the cytoskeleton and its associated motors have an important role in membrane remodelling. Actin and myosin 1 have been implicated in the invagination of the plasma membrane during endocytosis. However, whether myosin 1 and actin contribute to the membrane deformation that gives rise to the formation of post-Golgi carriers is unknown. Here we report that myosin 1b regulates the actin-dependent post-Golgi traffic of cargo, generates force that controls the assembly of F-actin foci and, together with the actin cytoskeleton, promotes the formation of tubules at the TGN. Our results provide evidence that actin and myosin 1 regulate organelle shape and uncover an important function for myosin 1b in the initiation of post-Golgi carrier formation by regulating actin assembly and remodelling TGN membranes.     

A comparison of the drying of chymotrypsin bound to bead cellulose by lyophilisation and in air at 25 to 70°C

Summary The effect was studied of the method of drying chymotrypsin attached to bead cellulose on its chemical and physical characteristics. These characteristics are not deteriorated when replacing the commonly used lyophilisation by fluid drying in the air stream. The preparations saved essentially the same proteolytic activity as well as stability even when increasing the drying air temperature to 70°C.     

Comparison of procainamide and 2-aminobenzamide labeling for profiling and identification of glycans by liquid chromatography with fluorescence detection coupled to electrospray ionization–mass spectrometry

One of the most widely used methods for glycan analysis is fluorescent labeling of released glycans followed by hydrophilic interaction chromatography–(ultra-)high-performance liquid chromatography [HILIC–(U)HPLC]. Here, we compare the data obtained by (U)HPLC–fluorescence (FLR) coupled to electrospray ionization–mass spectrometry (ESI–MS) for procainamide and 2-aminobenzamide (2-AB)-labeled N-glycans released from human immunoglobulin G (IgG). Fluorescence profiles from procainamide show comparable chromatographic separation to those obtained for 2-AB but gave higher fluorescence intensity as well as significantly improved ESI efficiency (up to 30 times that of 2-AB). Thus, labeling with procainamide increases the ability to identify minor glycan species that may have significant biological activity.     

Case study of combinatorial imaging: What protocol and what chlorophyll fluorescence image to use when visualizing infection of Arabidopsis thaliana by Pseudomonas syringae?

Localized infection of a plant can be mapped by a sequence of images capturing chlorophyll fluorescence transients in actinic light. Choice of the actinic light protocol co-determines fluorescence contrast between infected leaf segment and surrounding healthy tissue. Frequently, biology cannot predict with which irradiance protocol, in which fluorescence image of the sequence, and in which segment of the image there will be the highest contrast between the healthy and infected tissue. Here, we introduce a new technique that can be applied to identify the combination of chlorophyll fluorescence images yielding the highest contrast. The sets of the most contrasting images vary throughout the progress of the infection. Such specific image sets, stress-revealing fluorescence signatures, can be found for the initial and late phases of the infection. Using these signatures, images can be divided into segments that show tissue in different infection phases. We demonstrate the capacity of the algorithm in an investigation of infection of the model plant Arabidopsis thaliana by the bacterium Pseudomonas syringae. We show that the highest contrast is found with transients elicited by fluctuating, harmonically modulated irradiance with long periods.     

Identification of Histidine Residues Involved in Zn2+ Binding to αA- and αB-Crystallin by Chemical Modification and MALDI TOF Mass Spectrometry

??-Crystallin, a member of the small heat shock protein family is the major protein of mammalian eye lens and is a molecular chaperone. As there is no protein turn over in the lens, stability of ??-crystallin is one of the most crucial factors for its survival and function. We previously reported that the molecular chaperone-like activity and stability of ??-crystallin dramatically increased in the presence of Zn²⁺ (Biochemistry, 2008). We also reported that each subunit of ??-crystallin could bind multiple zinc ions through inter-subunit bridging giving rise to enhanced stability (Biopolymers, 2011). The amino acid residues involved in zinc binding were not known. Since cysteine residues were not responsible for binding to Zn²⁺, we tried to identify the histidine residues bound to zinc ions. We modified recombinant ??A- and ??B-crystallin with diethylpyrocarbonate (DEPC) a histidine modifying reagent, in presence and absence of Zn²⁺ followed by tryptic digestion. The residues modified by DEPC were identified through peptide mass matching by MALDI mass spectrometry. We have clearly identified H79, H107 and H115 of ??A-crystallin and H104, H111 and H119 of ??B-crystallin as the Zn²⁺ binding residues. The significance of the histidine rich sequence region of ??-crystallin for its stability is discussed.     

Identification and quantification of concentration-dependent biomarkers in MCF-7/BOS cells exposed to 17β-estradiol by 2-D DIGE and label-free proteomics

This paper reports the identification of biomarkers resulting from the exposure of MCF-7/BOS cells to 17β-estradiol (E(2)). The biomarkers were identified using 2 independent and complementary techniques, 2-D DIGE/MALDI-TOF peptide mass fingerprint, and 2-D UPLC-ESI MS/MS. They were identified from the cytosolic fractions of cells treated for 24h with mitogenic concentrations of 1, 30 and 500 pM of 17β-estradiol. Five biomarkers were up-regulated proteins, namely HSP 74, EF2, FKBP4, EF1 and GDIB and one was a down-regulated protein, namely K2C8. Three of these proteins, EF2, FKBP4 and K2C8 are implicated in a network centered on the estrogen receptors ESR1 and ESR2 as well as on AKT1. After the discovery phase, three biomarkers were selected to test the presence of estrogens using selected reaction monitoring (SRM). They were monitored using SRM after incubation of MCF-7/BOS in the presence of E(2) for confirmation or selected xenoestrogens. Daidzein, coumestrol and enterolactone induced an up-regulation of EF2 and FKPB4 proteins, while tamoxifen and resveratrol induced a down-regulation. The exposure of all phytoestrogens induced the down-regulation of K2C8. These markers form a preliminary molecular signature that can be used when testing the estrogenic activity of xenobiotics, either pure or in mixtures.     

Loss of Ssq1 leads to mitochondrial dysfunction,activation of autophagy and cell cycle arrest due to iron overload triggered by mitochondrial iron–sulfur cluster assembly defects in Candida albicans

Iron–sulfur clusters perform essential functions in enzymatic catalysis and homeostatic regulation. Here we for the first time identified Ssq1 as an essential component for iron–sulfur cluster assembly in Candida albicans. Ssq1 played an important role in cell growth. Shutting off SSQ1 led to accumulation of intracellular iron, especially in mitochondria, and disorder of intracellular iron regulation. In tetO-SSQ1, iron overloading triggered the oxidative damage of mitochondrial function. Surprisingly, disruption of SSQ1 activated autophagic pathway. The mitochondrial dysfunction was further aggravated when CCZ1 (which is essential for autophagy) and SSQ1 was simultaneously deleted, suggesting that autophagy played a critical role in maintenance of mitochondrial function in tetO-SSQ1. In addition, double deletion of SSQ1 and CCZ1 further elevated cellular iron levels in comparison with tetO-SSQ1, indicating that autophagy participated in maintenance of iron homeostasis. Furthermore, we found that loss of SSQ1 led to increasing protein expression of Rnr1 and redistribution of Rnr2 from the nucleus to cytoplasm, and further resulted in cell cycle arrest. The results implied that cell cycle arrest was caused by activating the checkpoint pathway because of impairing the iron–sulfur cluster assembly in tetO-SSQ1. Shutting off SSQ1 led to a significant defect in filamentous development. Interestingly, the tetO-SSQ1ccz1Δ/Δ growth was inhibited on hyphae-inducing solid media. Both tetO-SSQ1 and tetO-SSQ1ccz1Δ/Δ exhibited extremely attenuated virulence, indicating that Ssq1 might provide a promising target for antifungal drugs development. In summary, our findings provide new insights into the understanding of iron–sulfur cluster assembly-related gene in C. albicans.     

Is ability to hepatic steatosis influenced by age at the beginning of the overfeeding period in Muscovy and Pekin ducks?

The aim of this study was to analyse the effects of species (Muscovy and Pekin ducks) and age at the beginning of the overfeeding period on fatty liver production, carcass composition and lipid and moisture content of the liver and breast muscle. We reared four groups of 40 ducks per species for the study, starting at 2-week intervals in order to have four different ages together at the beginning of the overfeeding period (10, 12, 14 and 16 weeks). At the end of the overfeeding period, all ducks were slaughtered. Our results confirmed the high levels of difference in carcass composition and lipid content in the plasma, liver and breast muscle between Muscovy and Pekin ducks at all ages. Pekin ducks were not able to develop a high degree of hepatic steatosis, but had increased lipid storage in peripheral adipose and muscle tissues than Muscovy ducks. However, the fatty liver weight of Pekin ducks increased with age, with lipid deposition in the liver and peripheral tissues. The ability of Muscovy ducks to produce fatty livers remained unchanged with age in line, with lipid deposition in the liver and peripheral tissues. The sites of lipid deposition thus depend on species and not on the physiological maturity of ducks.     

Low-level laser therapy with 850 nm recovers salivary function via membrane redistribution of aquaporin 5 by reducing intracellular Ca2+ overload and ER stress during hyperglycemia

The overall goal is to study the effect of low-level laser therapy (LLLT) on membrane distribution of major water channel protein aquaporin 5 (AQP5) in salivary gland during hyperglycemia. Par C10 cells treated with high glucose (50?mM) showed a reduced membrane distribution of AQP5. The functional expression of AQP5 was downregulated due to intracellular Ca²⁺ overload and ER stress. This reduction in AQP5 expression impairs water permeability and therefore results in hypo-salivation. A reduced salivary flow was also observed in streptozotocin (STZ)-induced diabetic mice model and the expression of AQP5 and phospho-AQP5 was downregulated. Low-level laser treatment with 850?nm (30?mW, 10?min?=?18?J/cm²) reduced ER stress and recovered AQP5 membrane distribution via serine phosphorylation in the cells. In the STZ-induced diabetic mouse, LLLT with 850?nm (60?J/cm²) increased salivary flow and upregulated of AQP5 and p-AQP5. ER stress was also reduced via downregulation of caspase 12 and CHOP. In silico analysis confirmed that the serine 156 is one of the most favorable phosphorylation sites of AQP5 and may contribute to the stability of the protein. Therefore, this study suggests high glucose inhibits phosphorylation-dependent AQP5 membrane distribution. High glucose induces intracellular Ca²⁺ overload and ER stress that disrupt AQP5 functional expression. Low-level laser therapy with 850?nm improves salivary function by increasing AQP5 membrane distribution in hyperglycemia-induced hyposalivation.     

Identification and apical membrane localization of an electrogenic Na⁺/Ca²⁺ exchanger NCX2a likely to be involved in renal Ca²⁺ excretion by seawater fish

Seawater (SW) contains ～10 mM Ca(2+), yet marine fish must drink seawater as their major water source. Thus marine teleosts fish need to excrete Ca(2+) to maintain whole body Ca(2+) homeostasis. In the intestine, seawater Ca(2+) interreacts with epithelial-secreted HCO(3)(-) by the intestinal epithelium, and the resulting CaCO(3) precipitates, which is rectally excreted. Recently the transporters involved in intestinal HCO(3)(-) secretion were identified. Ca(2+) is also excreted by the kidney, but the protein(s) involved in renal Ca(2+) excretion have not been identified. Here we identified a candidate transporter by using SW pufferfish torafugu (Takifugu rubripes) and its closely related euryhaline species mefugu (Takifugu obscurus), which are becoming useful animal models for studying molecular mechanisms of seawater adaptation. RT-PCR analyses of Na(+)/Ca(2+) exchanger (NCX) family members in various torafugu tissues demonstrated that only NCX2a is highly expressed in the kidney. Renal expression of NCX2a was markedly elevated when mefugu were transferred from freshwater to seawater. In situ hybridization and immunohistochemical analyses indicated that NCX2a is expressed in the proximal tubule at the apical membrane. NCX2a, expressed in Xenopus oocytes, conferred [Ca(2+)](out)- and Na(+)-dependent currents. These results suggest that NCX2a mediates renal Ca(2+) secretion at the apical membrane of renal proximal tubules and has an important role in whole body Ca(2+) homeostasis of marine teleosts.     

Synergistic Induction of Endothelin-1 by Tumor Necrosis Factor �� and Interferon �� Is due to Enhanced NF-��B Binding and Histone Acetylation at Specific ��B Sites

Endothelin-1 (ET-1) is a potent vasoconstrictor and co-mitogen for vascular smooth muscle and is implicated in pulmonary vascular remodeling and the development of pulmonary arterial hypertension. Vascular smooth muscle is an important source of ET-1. Here we demonstrate synergistic induction of preproET-1 message RNA and release of mature peptide by a combination of tumor necrosis factor α (TNFα) and interferon γ (IFNγ) in primary human pulmonary artery smooth muscle cells. This induction was prevented by pretreatment with the histone acetyltransferase inhibitor anacardic acid. TNFα induced a rapid and prolonged pattern of nuclear factor (NF)-κB p65 subunit activation and binding to the native preproET-1 promoter. In contrast, IFNγ induced a delayed activation of interferon regulatory factor-1 without any effect on NF-κB p65 nuclear localization or consensus DNA binding. However, we found cooperative p65 binding and histone H4 acetylation at distinct κB sites in the preproET-1 promoter after stimulation with both TNFα and IFNγ. This was associated with enhanced recruitment of RNA polymerase II to the ATG start site and read-through of the ET-1 coding region. Understanding such mechanisms is crucial in determining the key control points in ET-1 release. This has particular relevance to developing novel treatments targeted at the inflammatory component of pulmonary vascular remodeling.Endothelin-1 is a 21-amino acid peptide which is known to be both a potent vasoconstrictor and mitogen for vascular smooth muscle (1, 2). It is released as a 38-amino acid precursor (Big ET-1²) before cleavage to the mature ET-1 form. As such it has been implicated in the pathogenesis of vascular disease and is particularly associated with pulmonary arterial hypertension (3). Indeed, several endothelin receptor antagonists are now approved for the treatment of pulmonary arterial hypertension (4). However, endothelin receptor antagonists as a class are associated with potentially serious side effects (4), making new treatments aimed at blocking ET-1 synthesis an attractive alternative.Although endothelial cells are thought to be the main source of ET-1 release, several groups including our own have shown that ET-1 can be released from the more numerous vascular smooth muscle cells (5–10). The vascular pathology observed in pulmonary arterial hypertension is propagated by inflammation, and circulating levels of cytokines including tumor necrosis factor α (TNFα) are elevated in patients with pulmonary arterial hypertension (11–15). In many cell types cytokines mediate their biological effects at least in part by the activation of the nuclear factor κB (NF-κB) pathway (16), and a role for NF-κB in pulmonary arterial hypertension has been proposed (17). In addition, we have shown previously that a combination of TNFα and interferon γ (IFNγ) stimulates human pulmonary artery smooth muscle (HPASM) cells to release ET-1 (18). However, the mechanisms underlying this effect are unknown.The preproET-1 promoter region has been shown experimentally to possess binding sites for nuclear factor (NF)-1 and phorbol ester-sensitive c-Fos and c-Jun complexes (19), acute phase reactant regulatory proteins, and binding sites for AP-1 and GATA-2 (20–22). In addition, binding sites for interferon regulatory factor-1 (IRF-1) and NF-κB are predicted by Transfac analysis (23). The close proximity of the IRF-1 site and one of the NF-κB sites is characteristic of genes that are regulated by the synergistic action of TNFα and IFNγ, such as interleukin-6 (IL-6) and intercellular adhesion molecule-1 (24, 25), although ET-1 has not previously been recognized in this group.Our aims were, therefore, to investigate the role of NF-κB in ET-1 release by primary HPASM cells. In addition, we were interested in the role of histone acetylation in the epigenetic control of the ET-1 production. Understanding these novel mechanisms will allow a greater understanding of the pathogenesis of vascular remodeling in pulmonary vessels and aid in the development of new treatment strategies aimed at blocking synthesis of ET-1.     

Activation of guanine-β-d-arabinofuranoside and deoxyguanosine to triphosphates by a common pathway blocks T lymphoblasts at different checkpoints

The deoxyguanosine (GdR) analog guanine-ß-d-arabinofuranoside (araG) has a specific toxicity for T lymphocytes. Also GdR is toxic for T lymphocytes, provided its degradation by purine nucleoside phosphorylase (PNP) is prevented, by genetic loss of PNP or by enzyme inhibitors. The toxicity of both nucleosides requires their phosphorylation to triphosphates, indicating involvement of DNA replication. In cultured cells we found by isotope-flow experiments with labeled araG a rapid accumulation and turnover of araG phosphates regulated by cytosolic and mitochondrial kinases and deoxynucleotidases. At equilibrium their partition between cytosol and mitochondria depended on the substrate saturation kinetics and cellular abundance of the kinases leading to higher araGTP concentrations in mitochondria. dGTP interfered with the allosteric regulation of ribonucleotide reduction, led to highly imbalanced dNTP pools with gradual inhibition of DNA synthesis and cell-cycle arrest at the G1-S boundary. AraGTP had no effect on ribonucleotide reduction. AraG was in minute amounts incorporated into nuclear DNA and stopped DNA synthesis arresting cells in S-phase. Both nucleosides eventually induced caspases and led to apoptosis. We used high, clinically relevant concentrations of araG, toxic for nuclear DNA synthesis. Our experiments do not exclude an effect on mitochondrial DNA at low araG concentrations when phosphorylation occurs mainly in mitochondria.