Role of Transglycosylation Products in the Expression of Multiple Xylanases in <Emphasis Type="Italic">Myceliophthora</Emphasis> sp. IMI 387099

This study reports the regulation of multiple xylanases produced by Myceliophthora sp. IMI 387099. Fructose was found to positively regulate the expression of multiple xylanase when used as sole carbon source. The xylanases (EX₁ and EX₂) of acidic pI were expressed in the presence of simple sugars (glucose, arabinose, and xylose), whereas xylanase of both acidic as well as basic pI (EX_1, EX_2, EX₃, and EX₅) were expressed in the presence of fructose, xylan, and combination of xylan and alcohol. The combination of fructose and xylan also led to expression of an additional xylanase (EX₄). The positional isomer (iso-X4) was found to be the key transglycosylation product when cultures were grown in the presence of fructose and xylan. In the presence of alcohols, the higher expression of xylanase was ascribed to the synergistic effect of alkyl glycoside and other transglycosylation products present in the culture extracts.     

Exogenous glycine betaine modulates ascorbate peroxidase and catalase activities and prevent lipid peroxidation in mild water-stressed Carapa guianensis plants

The hypothesis that application of exogenous glycine betaine (GB_EX) may attenuate the effects of mild water deficit in leaf gas exchange and lipid peroxidation in Carapa guianensis was examined. For this reason, 110-d old plants were sprayed with 0, 25, and 50 mM GB_EX and then subjected to two watering regimes. In the first, irrigation was continuously performed to maintain the soil near to field capacity (watered plants). In the second, irrigation was withheld and water deficit resulted from progressive evapotranspiration (water-stressed plants). Treatment comparisons were assessed when predawn leaflet water potential (Ψ_pd) of stressed plants reached ?1.28 ± 0.34 MPa. Regardless of the watering regime, significant (P<0.05) increases in foliar glycine betaine (GB_Leaf) concentration were observed in response to increasing GB_EX; however, such increases were more expressive in stressed plants. The net photosynthetic rate, stomatal conductance to water vapor, and intercellular to ambient CO₂ concentration ratio were significantly lower in water-stressed plants independently of GB_EX concentration sprayed on leaves. The application of 25 and 50 mM GB_EX caused significant (P<0.05) increases in ascorbate peroxidase (APX) activity in stressed plants, while significant (P<0.05) increases in catalase activity was observed just in the stressed plants treated with 50 mM GB_EX. Malondialdehyde concentrations did not differ between watered and stressed plants regardless of GBEX concentration. In conclusion, C. guianensis was able to incorporate GBEX through their leaves and the resulting increases in GB_Leaf attenuated lipid peroxidation in stressed plants through positive modulation of APX and CAT activities.     

Hydrogen exchange of the tryptophan residues in bovine alpha-lactalbumin studied by UV spectroscopy

The effect of Ca²⁺ ion on structural fluctuation of a milk Ca²⁺-binding protein, α-lactalbumin, under native conditions was investigated by comparing hydrogen-exchange reactions of tryptophan residues in the apo-form without Ca²⁺ and in the holo-form at 1 mM CaCl₂ at pH 7.0 in the presence of 0.1M Na⁺. The reactions were followed by measuring time-dependent absorption changes at 298–300 nm due to the ²H-¹H exchange of the tryptophan imino protons and were found to be biphasic under all the conditions examined. Two of the four tryptophan protons are insensitive to Ca²⁺ concentration and show a relatively fast exchange rate. The other two protons are much more extensively protected (a protection degree of 10³–10⁵) and are markedly affected by the presence of Ca²⁺. Examinations of the temperature dependence and pH dependence of the individual exchange rates have been utilized for elucidating the exchange mechanism. The fast protons show a low activation energy reaction with so-called EX₂ kinetics. The exchange reaction of the slow protons is accompanied by a high activation energy, and the exchange mechanism of the protons depended on the presence or absence of stabilizing Ca²⁺ ions—the EX₁ kinetics for the apo-protein and the EX₂ kinetics for the holo-protein at 1 mM Ca²⁺. The exchange reaction in the thermally unfolded state was also found to be biphasic, but the fast phase, which has an exchange rate in the fully exposed state, becomes predominant with decreasing temperature. By taking this fact and using a structural unfolding model of hydrogen exchange, the present results are fully consistent with thermodynamic parameters of the thermal transition and kinetic parameters of refolding reactions induced by concentration jumps of guanidine hydrochloride obtained in previous studies. It is demonstrated that the reaction of the slow protons in the native state is mediated by a transient global unfolding equivalent to the “thermal” unfolding under a native condition and that switching of the exchange mechanism from the EX₁ to EX₂ kinetics results from acceleration of the refolding rate with an increase in Ca²⁺ concentration. The transient global unfolding takes place even under a strongly native condition, e.g., at a temperature 20° below the beginning of the thermal transition.     

From gallium to thallium: Group 13 one-dimensional polymers

The reactions of EX₃ (E = Ga, In, Tl, X = Br or Cl) with pyrazine and pyrazine, 2-carboxylic acid (pyzca-H) affords a series of one-dimensional ladder polymers, [EX₃(pyz)]_∞ (E = Ga, In, Tl, X = Cl, Br) and the zigzag polymer [InBr₂(OH₂)₂(pyzca)]_∞. These polymers are prepared in high yield through a facile synthesis. The structures of each were characterized in the solid state by single crystal X-ray analysis, infrared spectroscopy and where possible NMR. Experimental results reveal how stability of the one-dimensional polymer increases from gallium to thallium, from the air-sensitive gallium polymer to the aqueous preparation and air-stable products of indium and thallium.     

A general multistate model for the analysis of hydrogen-exchange kinetics

In proton nmr, the chemical exchange rates of slowly exchanging labile hydrogens (with lifetimes in the range ～ 10 msec – ～ 1 sec) of peptides, proteins, and nucleic acids can be measured in H₂O by a combination of two separate experiments: (1) the transfer of solvent saturation and (2) saturation-recovery experiments. When these molecules exist in a dynamic equilibrium among different conformations, the experiments cannot be analyzed in a straightforward manner to derive the intrinsic exchange rates. In the present study we have derived analytical expressions for the above two experiments on a biomolecule under certain limiting conditions: (1) the extreme low-motility limit, where each of the conformational transitions is much slower than the corresponding hydrogen exchange rate with the solvent; (2) the high-motility limit (EX₂ mechanism), which is the opposite extreme of the previous limit; and (3) the low-motility limit (EX₁ mechanism), which is a mixture of limits (1) and (2), i.e., for some of the conformations, the exchange rate with the solvent is much faster than their conformational transition rates, while for the remaining conformations the reverse situation is realized. The results may be considered as a generalization to an arbitrary number of states of the two-state model treated by Hvidt. Equations have also been derived that are applicable to the iostope exchange method of measuring very slow exchange rates (with life-times of the order of minutes and longer) in biomolecules. The saturation recovery experiments performed in H₂O on the active pentapeptide fragment of thymopoietin serve to illustrate the high-motility limit. The theoretical formulation presented in this study can be easily adapted to other double-resonance techniques and also to situations where the kinetics of an arbitrary system existing in a multistate equilibrium are of interest.     

Evaluating UV-B effects and EDU protection in cucumber leaves using fluorescence images and fluorescence emission spectra

A newly developed laboratory fluorescence imaging system was used to obtain fluorescence images (FImage) of freshly excised cucumber (Cucumis sativus L.) leaves in spectral bands centered in the blue (F450), green (F550), red (F680), and far-red (F730) spectral regions that resulted from a broad-band (300-400 nm) excitation source centered at 360 nm. Means of relative fluorescence intensities (RFI) from these spectral fluorescence images were compared with spectral fluorescence emission data obtained from excitation wavelengths at 280 nm (280EX, 300-550 nm) and 380 nm (380EX, 400-800 nm) of dimethyl sulfoxide (DMSO) extracts from these leaves. All three fluorescence data types (FImage, 280EX, 380EX) were used to assess ultraviolet-B (UV-B, 280-320 nm) induced physiological changes and the possible use of N-[2-(2-oxo-1-imidazolidinyl) ethyl]-N′-phenylurea (EDU or ethylenediurea) as a chemical protectant against UV-B damage. Plants exhibited well known foliar growth and pigment responses to UV-B exposure (e.g., increased UV-B absorbing compounds and decreased leaf area, chlorophyll a content; and and lower chlorophyll a/b and chlorophyll/carotenoid pigment ratios). Since EDU alone had no effect on foliar variables, there was no evidence that EDU afforded protection against UV-B. Instead, EDU augmented some UV-B effects when provided in conjunction with UV-B irradiation (e.g., reductions in the chlorophyll/carotenoid ratio, total photosynthetic pigments, and chlorophyll b content).Relative fluorescence intensities (RFI) in the longer visible wavelengths (green, red, and far-red) were uncorrelated for comparisons between the FImage and 380EX data sets. However, blue and green RFI were significantly correlated (0.8r0.6; P ≤0.002) for comparisons between FImage and 280EX data sets. UV-B treatment caused an increase in blue RFI (e.g., F450) in both images and 280EX measurements. One explanation is that the UV-B excitation of both 280EX and FImage stimulates processes that produce excess blue fluorescence. The molecules that produce the excess blue fluorescence in both the 280EX and the Fimage data are different electron transfer agents that operate in parallel. For FImage, the UV excitation penetrates leaf surface layers to stimulate fluorescence from compounds in mesophyll and epidermal tissues (as occurs for the extracts of leaf discs), whereas emissions captured at longer, less energetic wavelengths, were primarily from the epidermal layer. UV-B irradiated leaves showed much greater heteorgeneity of RFI in both the green (F550_FImag) and the red (F680_FImag) bands than unirradiated leaves; this was true irrespective of EDU treatment.Although qualitative responses in individual bands differed between FImage and 380EX data, similar results were obtained in the detection of UV-B induced effects when the red/green and blue/far-red fluorescence ratios of these data were compared. The red/green ratio (either F680/F550_FImage or F675/F525_380EX) was lower for UV-B exposed plants in both images and 380EX data. UV-B exposure also significantly enhanced the blue/far-red ratio of images (F450/F740_FImage) and the comparable 380EX ratio (F450/F730_380EX) for the combined UV-B/EDU group. The far-red/red ratios were not useful in separating treatment effects in images or 380EX. Although comparable ratios were not available in 280EX data, the UV/blue ratio (F315/F420_280EX) was substantially reduced by UV-B exposure and was inversely related to total photosynthetic pigment content. These findings suggest that the red/green ratio (FImage, 380EX) and the UV/blue ratio (280EX) may be as useful as the blue/far-red ratio (380EX) reported previously in detection of UV-B stress. Furthermore, the results support the validity of the imaging technique as a non-destructive diagnostic tool for assessing UV-B stress damage in plants.     

Post-ExSELEX stabilization of an unnatural-base DNA aptamer targeting VEGF165 toward pharmaceutical applications

A new technology, genetic alphabet expansion using artificial bases (unnatural bases), has created high-affinity DNA ligands (aptamers) that specifically bind to target proteins by ExSELEX (genetic alphabet Expansion for Systematic Evolution of Ligands by EXponential enrichment). We recently found that the unnatural-base DNA aptamers can be stabilized against nucleases, by introducing an extraordinarily stable, unique hairpin DNA (mini-hairpin DNA) and by reinforcing the stem region with G–C pairs. Here, to establish this aptamer generation method, we examined the stabilization of a high-affinity anti-VEGF₁₆₅ unnatural-base DNA aptamer. The stabilized aptamers displayed significantly increased thermal and nuclease stabilities, and furthermore, exhibited higher affinity to the target. As compared to the well-known anti-VEGF₁₆₅ RNA aptamer, pegaptanib (Macugen), our aptamers did not require calcium ions for binding to VEGF₁₆₅. Biological experiments using cultured cells revealed that our stabilized aptamers efficiently inhibited the interaction between VEGF₁₆₅ and its receptor, with the same or slightly higher efficiency than that of the pegaptanib RNA aptamer. The development of cost-effective and calcium ion-independent high-affinity anti-VEGF₁₆₅ DNA aptamers encourages further progress in diagnostic and therapeutic applications. In addition, the stabilization process provided additional information about the key elements required for aptamer binding to VEGF₁₆₅.     

Identification of structural and molecular determinants of the tyrosine‐kinase Wzc and implications in capsular polysaccharide export

Capsular polysaccharides are well‐established virulence factors of pathogenic bacteria. Their biosynthesis and export are regulated within the transmembrane polysaccharide assembly machinery by the autophosphorylation of atypical tyrosine‐kinases, named BY‐kinases. However, the accurate functioning of these tyrosine‐kinases remains unknown. Here, we report the crystal structure of the non‐phosphorylated cytoplasmic domain of the tyrosine‐kinase Wzc from Escherichia coli in complex with ADP showing that it forms a ring‐shaped octamer. Mutational analysis demonstrates that a conserved EX₂RX₂R motif involved in subunit interactions is essential for polysaccharide export. We also elucidate the role of a putative internal regulatory tyrosine and we show that BY‐kinases from proteobacteria autophosphorylate on their C‐terminal tyrosine cluster via a single‐step intermolecular mechanism. This structure‐function analysis also allows us to demonstrate that two different parts of a conserved basic region called the RK‐cluster are essential for polysaccharide export and for kinase activity respectively. Based on these data, we revisit the dichotomy made between BY‐kinases from proteobacteria and firmicutes and we propose a unique process of oligomerization and phosphorylation. We also reassess the function of BY‐kinases in the capsular polysaccharide assembly machinery.     

Editorial

Interspecific interactions between the shrews Sorex araneus and S. minutus F. J. M. ELLENBROEK Noordbrabants Natuurmuseum, Postbus g24, 5000AX Tilburg, Netherlands Foraging in the European Hedgehog, Erinaceus europaeus ANDREW J. WROOT Department of Zoology, Royal Holloway College (University of London), Egham, Surrey TW20 0EX     

JAMM: a metalloprotease-like zinc site in the proteasome and signalosome

The JAMM (JAB1/MPN/Mov34 metalloenzyme) motif in Rpn11 and Csn5 underlies isopeptidase activities intrinsic to the proteasome and signalosome, respectively. We show here that the archaebacterial protein AfJAMM possesses the key features of a zinc metalloprotease, yet with a distinct fold. The histidine and aspartic acid of the conserved EX_nHS/THX₇SXXD motif coordinate a zinc, whereas the glutamic acid hydrogen-bonds an aqua ligand. By analogy to the active site of thermolysin, we predict that the glutamic acid serves as an acid-base catalyst and the second serine stabilizes a tetrahedral intermediate. Mutagenesis of Csn5 confirms these residues are required for Nedd8 isopeptidase activity. The active site-like architecture specified by the JAMM motif motivates structure-based approaches to the study of JAMM domain proteins and the development of therapeutic proteasome and signalosome inhibitors.     

Humus stock degradation and its impact on phosphorus forms in arable soils – a case of the Ukrainian Forest-Steppe Zone

Soil humus degradation strengthens nutrient mining, especially phosphorus. This study was carried out on the Ukrainian Forest-Steppe Zone (UFSZ). A total of 21 soil profiles have been investigated: 11 Phaeozems, 6 Luvisols, and 4 Chernozems. Soils were tested for particle size distribution, calcium carbonate (CC), pH, and organic carbon (C_org). The evaluation of humus degradation as a reason of P depletion was performed based on indicators such as Humus Stock Gap (HSG), Humus Stability Index (S), and Yield Gap/Gain (YG/G). In order to evaluate the degree of P depletion, total phosphorus (P_tot) and its five fractions: water soluble – P_H2O, exchangeable – P_EX, bound to Fe and Al – P_Fe/Al, bound to Ca – P_Ca, and residual P – Pres, have been determined.

Data revealed that in 14 of 21 investigated soils, S indices were below the threshold its value of nine, considered as the balanced content of humus with respect to soil texture. Next, in 11 of 21 cases, the negative humus balance indicates the yield gap in Phaeozems and Chernozems. The first three P pools (P_H2O, P­EX and P_Al/Fe) in Phaeozems were exhausted, constituting less than 10% of the P_tot. In Phaeozems, P_Al/Fe, in Luvisols, P_EX, and in Chernozems, P_H2O fractions were basic indicators of available P status. Their pools were directly or indirectly controlled by PCa. Humus content in Phaeozems and Chernozems revealed as the key factor impacting both total P and/or its available resources. Amelioration of P depletion requires efforts oriented on restoration of soil humus stock, concomitant with P fertilization.     

SDA,a DNA Aptamer Inhibiting E- and P-Selectin Mediated Adhesion of Cancer and Leukemia Cells,the First and Pivotal Step in Transendothelial Migration during Metastasis Formation

Endothelial (E-) and platelet (P-) selectin mediated adhesion of tumor cells to vascular endothelium is a pivotal step of hematogenous metastasis formation. Recent studies have demonstrated that selectin deficiency significantly reduces metastasis formation in vivo. We selected an E- and P-Selectin specific DNA Aptamer (SDA) via SELEX (Systematic Evolution of Ligands by EXponential enrichment) with a K _d value of approximately 100 nM and the capability of inhibiting the interaction between selectin and its ligands. Employing human colorectal cancer (HT29) and leukemia (EOL-1) cell lines we could demonstrate an anti-adhesive effect for SDA in vitro. Under physiological shear stress conditions in a laminar flow adhesion assay, SDA inhibited dynamic tumor cell adhesion to immobilized E- or P-selectin. The stability of SDA for more than two hours allowed its application in cell-cell adhesion assays in cell culture medium. When adhesion of HT29 cells to TNFα-stimulated E-selectin presenting human pulmonary microvascular endothelial cells was analyzed, inhibition via SDA could be demonstrated as well. In conclusion, SDA is a potential new therapeutic agent that antagonizes selectin-mediated adhesion during metastasis formation in human malignancies.     

Discriminating mild from critical COVID-19 by innate and adaptive immune single-cell profiling of bronchoalveolar lavages

How the innate and adaptive host immune system miscommunicate to worsen COVID-19 immunopathology has not been fully elucidated. Here, we perform single-cell deep-immune profiling of bronchoalveolar lavage (BAL) samples from 5 patients with mild and 26 with critical COVID-19 in comparison to BALs from non-COVID-19 pneumonia and normal lung. We use pseudotime inference to build T-cell and monocyte-to-macrophage trajectories and model gene expression changes along them. In mild COVID-19, CD8⁺ resident-memory (T_RM) and CD4⁺ T-helper-17 (T_H17) cells undergo active (presumably antigen-driven) expansion towards the end of the trajectory, and are characterized by good effector functions, while in critical COVID-19 they remain more naïve. Vice versa, CD4⁺ T-cells with T-helper-1 characteristics (T_H1-like) and CD8⁺ T-cells expressing exhaustion markers (T_EX-like) are enriched halfway their trajectories in mild COVID-19, where they also exhibit good effector functions, while in critical COVID-19 they show evidence of inflammation-associated stress at the end of their trajectories. Monocyte-to-macrophage trajectories show that chronic hyperinflammatory monocytes are enriched in critical COVID-19, while alveolar macrophages, otherwise characterized by anti-inflammatory and antigen-presenting characteristics, are depleted. In critical COVID-19, monocytes contribute to an ATP-purinergic signaling-inflammasome footprint that could enable COVID-19 associated fibrosis and worsen disease-severity. Finally, viral RNA-tracking reveals infected lung epithelial cells, and a significant proportion of neutrophils and macrophages that are involved in viral clearance.Subject terms: Genome-wide analysis of gene expression, Innate immunity, Bioinformatics     

Microbial Biomass-C in Reclaimed Soil of the Rhineland (Germany) and the North Bohemian Lignite Mining Areas (Czech Republic): Measured and Predicted Values

This article compares reclaimed soils in the Rhineland lignite mining area, Germany (RA) and in the north Bohemian brown coal basin, Czech Republic (NBB). Because of highly significant differences of physical and chemical characteristics (RA was characterized by lower content of sand, clay, soil organic carbon, and total nitrogen) between both analyzed areas, the content of microbial biomass carbon (C_MB) differs significantly (p < 0.01): RA: 241.17 ± 96.50 μg C/g dry soil; NBB: 424.42 ± 136.23 μg C/g. Extracellular microbial carbon (C_EX) was also significantly higher in RA than NBB. The measured C_MB values from both mining areas were verified using two empirical mathematical models. Because our former model (used for evaluation of NBB) did not fit soils with low organic carbon content (0.10–0.80%), a new model was proposed. For evaluation of RA reclaimed sites the measured, model‐predicted, and calculated values were used. The best site in RA was Dürwiss Deponie (site 18 near Eschweiler), reclaimed in 1965 and characterized by good soil physical parameters (sand 4%, silt 77%, and clay 19%). In NBB undisturbed soils were the best, together with the site of Ú?ín (site 2 near Ústí and Labem), reclaimed in 1964, where high‐quality reclamation management resulted in high biological activity. According to biological criteria, the best management practice in NBB was use of high‐quality loam overlaying montmorillonite clayey subsoil. In RA direct reclamation of high‐quality loess with low content of sand also gave good results.     

Loss of the circadian rhythms of locomotor activity, food intake, and plasma melatonin concentration induced by constant bright light in the pigeon (Columba livia)

Summary Locomotor activity and feeding activity were measured together with circulating levels of melatonin in pigeons which were exposed to constant bright light (LLbright, 2000 lux) following light-dark (LD) cycles. Although all the pigeons showed daily rhythms of locomotor activity, feeding activity, and melatonin levels under LD cycles, they lost all the rhythms in prolonged LLbright. Acute exposure to bright light (2000 lux) during darkness reduced plasma melatonin levels. The half-time for the suppression in melatonin levels was about 30 min after short-term light exposure. These results support the hypothesis that melatonin may control the circadian rhythms of locomotor activity and feeding activity in the pigeon.Abbreviations LD light-dark - LLdim constant dim light - LLbright constant bright light - DD constant darkness - PX pinealectomy - EX blinding - RIA radioimmunoassay     

Structural similarity and functional diversity in diiron-oxo proteins

 Diiron-oxo proteins currently represent one of the most rapidly developing areas of bioinorganic chemistry. All of these proteins contain a four-helix bundle protein fold surrounding a (μ-carboxylato)diiron core, and most, if not all, of the diiron(II) sites appear to react with O₂ as part of their functional processes. Despite these common characteristics, an emerging functional diversity is one of the most striking aspects of this class of proteins. X-ray crystal structures of diiron(II) sites are now available for four of these proteins: hemerythrin (Hr), the hydroxylase protein of methane monooxygenase (MMOH), the R2 protein of Escherichia coli ribonucleotide reductase (RNR-R2), and a plant acyl-carrier protein Δ⁹-desaturase. The structure of the diiron(II) site in Hr, the sole O₂ carrier in the group, is clearly distinct from the other three, whose function is oxygen activation. The Hr diiron site is more histidine rich, and the oxygen-activating diiron sites contain a pair of (D/E)X_30–37EX₂H ligand sequence motifs, which is clearly not found in Hr. The Hr diiron site apparently permits only terminal O₂ coordination to a single iron, whereas the oxygen-activating diiron(II) centers present open or labile coordination sites on both irons of the center, and show a much greater coordinative flexibility upon oxidation to the diiron(III) state. Intermediates at the formal Fe^IIIFe^III and Fe^IVFe^IV oxidation levels for MMOH and formal Fe^IIIFe^IV oxidation level for RNR-R2 have been identified during reactions of the diiron(II) sites with O₂. An [Fe₂(μ-O)₂]^4+, 3+ "diamond core" structure has been proposed for the latter two oxidation levels. The intermediate at the Fe^IIIFe^IV oxidation level in RNR-R2 is kinetically competent to generate a stable, functionally essential tyrosyl radical. The Fe^IVFe^IV oxidation level is presumed to effect hydroxylation of hydrocarbons in MMOH, but the mechanism of this hydroxylation, particularly the involvement of discrete radicals, is currently controversial. The biological function of diiron sites in three members of this class, rubrerythrin, ferritin and bacterioferritin, remains enigmatic. Received: 31 July 1996 / Accepted: 4 October 1996     

Identification and biochemical characterization of WbwB,a novel UDP-Gal: Neu5Ac-R α1,4-galactosyltransferase from the intestinal pathogen <Emphasis Type="Italic">Escherichia coli</Emphasis> serotype O104

The intestinal pathogen Escherichia coli serotype O104:H4 (ECO104) can cause bloody diarrhea and haemolytic uremic syndrome. The ECO104 O antigen has the unique repeating unit structure [4Galα1–4Neu5,7,9Ac₃α2–3Galβ1–3GalNAcβ1-], which includes the mammalian sialyl-T antigen as an internal structure. Previously, we identified WbwC from ECO104 as the β3Gal-transferase that synthesizes the T antigen, and showed that α3-sialyl-transferase WbwA transfers sialic acid to the T antigen. Here we identify the wbwB gene product as a unique α1,4-Gal-transferase WbwB that transfers Gal from UDP-Gal to the terminal sialic acid residue of Neu5Acα2–3Galβ1–3GalNAcα-diphosphate-lipid acceptor. NMR analysis of the WbwB enzyme reaction product indicated that Galα1-4Neu5Acα2–3Galβ1–3GalNAcα-diphosphate-lipid was synthesized. WbwB from ECO104 has a unique acceptor specificity for terminal sialic acid as well as the diphosphate group in the acceptor. The characterization studies showed that WbwB does not require divalent metal ion as a cofactor. Mutagenesis identified Lys243 within an RKR motif and both Glu315 and Glu323 of the fourth EX₇E motif as essential for the activity. WbwB is the final glycosyltransferase in the biosynthesis pathway of the ECO104 antigen repeating unit. This work contributes to knowledge of the biosynthesis of bacterial virulence factors.     

C-Terminus of Apolipoprotein A-I Removes Phospholipids from a Triolein/Phospholipids/Water Interface, but the N-Terminus Does Not: A Possible Mechanism for Nascent HDL Assembly

Apolipoprotein A-I (ApoA-I) is the principle protein component of HDL, also known as “good cholesterol,” which is an inverse marker for cardiovascular disease. The N-terminal 44 amino acids of ApoA-I (N44) are predicted to be responsible for stabilization of soluble ApoA-I, whereas the C-terminal 46 amino acids (C46) are predicted to initiate lipid binding and oligomerization. In this work, we apply what we believe to be a novel application of drop tensiometry to study the adsorption and desorption of N44 and C46 at a triolein/POPC/water (TO/POPC/W) interface. The amount of peptide that adsorbed to the surface was dependent on the surface concentration of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and pressure (Π) before adsorption. At a TO/POPC/W interface, the exclusion pressure (Π_EX) of C46 was 25.8 mN/m, and was 19.3 mN/m for N44. Once adsorbed, both peptides formed a homogeneous surface with POPC but were progressively ejected from the surface by compression. During a compression, C46 removed POPC from the surface whereas N44 did not. Repeated compressions caused C46 to deplete entirely the surface of phospholipid. If full-length ApoA-I could also remove phospholipid, this could provide a mechanism for the transfer of surface components of chylomicrons and very low density lipoprotein to high density lipoprotein with the assistance of phospholipid transfer protein.     

Pineal and retinal melatonin is involved in the control of circadian locomotor activity and body temperature rhythms in the pigeon

Summary Although pinealectomy or blinding resulted in loss of the clarity of the free-running rhythm of locomotor activity and body temperature and reduced the peak level of circulating melatonin rhythms to approximately a half in intact pigeons, neither pinealectomy nor blinding abolished any of these rhythms. However, when pinealectomy and blinding were combined, the rhythms of locomotor activity and body temperature disappeared in prolonged constant dim light, and melatonin concentration was reduced to the minimum level of detection. In order to examine the role of melatonin in the pigeon's circadian system, it was administered either daily or continuously to PX + EX-pigeons in LLdim. Daily administration of melatonin restored circadian rhythms of locomotor activity which entrained to melatonin injections, but continuous administration did not induce any remarkable change of locomotor activity. These results suggest that melatonin synthesized in the pineal body and the eye contributes to circulating melatonin and its rhythmicity is important for the control of circadian rhythms of locomotor activity and body temperature in the pigeon.Abbreviations LD Light-dark - LLdim constant dim light - LLbright constant bright light - PX pinealectomy - EX blinding - SCN suprachiasmatic nucleus