Synthesis of the tri- and tetra-saccharides related to the fine structures of lichenan and cereal β-d-glucans

Syntheses, based on silver trifluoromethanesulfonate-promoted Koenigs-Knorr type condensations, are described of the d-glucotrioses, β-d-Glcp-(1→3)-β-d-Glcp-(1→4)-d-Glcp and β-d-Glcp-(1→4)-β-d-Glcp-(1→3)-d-Glcp, and the d-Glucotetraoses, β-d-Glcp-(1→3)-β-d-Glcp-(1→4)-β-d-Glcp-(1→4)-d-Glcp, β-d-Glcp-(1→4)-β-d-Glcp-(1→3)-β-d-Glcp-(1→4)-d-Glcp, and β-d-Glcp-(1→4)-β-d-Glcp-(1→4)-β-d-Glcp-(1→3)-d-Glcp, corresponding to the tri- and tetra-saccharide units in the linear chains of (1→4)- and (1→3)-linked β-d-glucopyranosyl residues of lichenan, and of oat and barley β-d-glucans.     

How to identify and quantify the members of the Bacillus genus?

Members of the Bacillus genus are widely distributed throughout natural environments and have been studied for decades among others for their physiology, genetics, ecological functions, and applications. However, despite its prevalence in nature, the characterization and classification of Bacillus remain challenging due to its complex and ever-evolving taxonomic framework. This review addresses the current state of the Bacillus taxonomic landscape and summarizes the critical points in the development of Bacillus phylogeny. With a clear view of Bacillus phylogeny as a foundation, we subsequently review the methodologies applied in identifying and quantifying Bacillus, while also discussing their respective advantages and disadvantages.     

Utilization of the guanylyltransferase and methyltransferases of vaccinia virus to modify and identify the 5′-terminals of heterologous RNA species

Enzymes extracted from purified vaccinia virus particles were used to catalyze the guanylylation (i.e. capping) and/or methylation of heterologous RNA species containing two or three phosphates or the structure m⁷G(5′)pppN at their 5′-terminals. This procedure provides a novel and specific method of labeling the 5′-terminals with $\text{[}{}^{\mathrm{\alpha -32}}\text{P]GTP}$ or S-adenosyl-[methyl-³H]methionine. Analysis of the RNAs of satellite tobacco necrosis virus and tobacco mosaic virus that were modified in this manner indicated that the original 5′-terminal sequences were (p)ppApGpPy and m⁷G(5′)pppGpU, respectively, which were enzymatically converted to m⁷G(5′)pppA^mpGpPy and m⁷G(5′)pppG^mpU.     

Pathogenicity of ‘take-all’ fungus to oats: Its relationship to the concentration and detoxification of the four avenacins

Data for inhibition of the growth of Gaeumannomyces graminis var. tritici (Ggt) and var. avenae (Gga), Phialophora radicicola and Fusarium avenaceum, caused by avenacins, are presented. The avenacins found in all oat species examined are sufficient in quantity to totally suppress growth of wheat ‘take-all’ (Ggt), even old roots containing 25 μg/g (fr. wt). Fungal variants that can also attack oats [var. avenae (Gga)] show considerable variations in their tolerance to avenacin A-1, ec₅₀ values being 5–80 μg/ml. Nevertheless, all Gga isolates maintained some growth at avenacin A-1 concentrations as high as 200 μg/ml and it is this ability to grow, albeit slowly, at high concentrations that is the critical difference between Gga and Ggt strains. The pathogenicity towards oats of a range of isolates of Gga is related to the fungicidal activity of avenacins. Gga pathogenicity is shown to increase with poor nutrition of the oat hosts (poor illumination, lack of minerals). Fungal detoxification of avenacins produces mono-deglucosylavenacin A-1, bis-deglucosylavenacin A-1 and, in one case, tris-deglycosylavenacin A-1. Ggt strains left avenacin A-1 almost unaffected giving only traces of mono-deglucosyl product. Gga strains bring about mono- and bis-deglucosylation whilst Fusarium avenaceum causes mainly bis-deglucosylation. Mono-deglucosylavenacin is shown to be less inhibitory to Gga than is avenacin A-1, whilst the bis-deglucosyl compound is still less inhibitory.     

Long-distance ABA Signaling and Its Relation to Other Signaling Pathways in the Detection of Soil Drying and the Mediation of the Plant’s Response to Drought

In this article we review evidence for a variety of long-distance signaling pathways involving hormones and nutrient ions moving in the xylem sap. We argue that ABA has a central role to play, at least in root-to-shoot drought stress signaling and the regulation of functioning, growth, and development of plants in drying soil. We also stress the importance of changes in the pH of the leaf cell apoplast as influenced both by edaphic and climatic variation, as a regulator of shoot growth and functioning, and we show how changes in xylem and apoplastic pH can affect the way in which ABA regulates stomatal behavior and growth. The sensitivity to drought of the pH/ABA sensing and signaling mechanism is emphasized. This allows regulation of plant growth, development and functioning, and particularly shoot water status, as distinct from stress lesions in growth and other processes as a reaction to perturbations such as soil drying.     

Safety of the production process of SURFACEN® to inactivate and remove virus

SURFACEN^® is a biological product produced from pig lungs. Since these animals can be potential sources of microbial pathogens such as viruses, the manufacturing process of this product should guarantee safety from health hazards. The SURFACEN^® production procedure is capable of effective viral clearance (inactivation/removal) by involving two stages of organic solvent extraction followed by acetone precipitation and heat treatment. In this study, we evaluated the clearance capacity of these four stages for a wide range of viruses by performing spiking experiments. Residual contamination was assessed using a Tissue Culture Infectious Dose assay (log₁₀ TCID₅₀). The validation study demonstrated that, for all viruses tested, the TCID₅₀ titers were reduced by more than 2 log₁₀ in each stage. Total log reduction values achieved were between ≥17.82 log₁₀ and ≥27.93 log₁₀, depending on the virus physical properties, titer, and the number of processing stages applied. Results indicated that the production procedure of SURFACEN^® can inactivate or remove contaminant viruses from the raw material.     

Reaction modelling and simulation to assess the integrated use of transketolase and ω-transaminase for the synthesis of an aminotriol

The most attractive, as well as challenging, multistep organic syntheses would preferably be carried out in a single reactor, as a one-pot synthesis. For biocatalytic syntheses, multistep reactions in one-pot mode bring a number of advantages, while at the same time raising unique challenges such as the compatibility of different biocatalysts. In this paper, we have developed a transketolase–transaminase (TK-TAm) two-step one-pot aminotriol synthesis reaction model, which integrates reaction kinetic models with process characterization (consisting of component degradation as a function of pH and concentration, aldehyde toxicity towards the enzyme, and ketol donor and acceptor side-reactions with TAm). Based on the analysis of the effect of the TAm/TK activity ratio on product yield, simulations provided guidance for further process and biocatalyst development.     

Molecular Clocks and the Timing of the Placental and Marsupial Radiations in Relation to the Cretaceous–Tertiary Boundary

Paleostratigraphic estimates of divergence time for nine independent cladogenic events within Mammalia, ranging from 14 to 130 million years, were regressed against Tamura–Nei-corrected 12S rRNA transversions. Relative rate-adjusted distances were also regressed against paleostratigraphic divergence times. The resulting equations were used to estimate interordinal divergence times within Eutheria and Metatheria for a data set that includes representatives of all orders in each infraclass. Without the adjustment for rate variation, divergence times range from 34 to 156 million years for placental orders, versus 32 to 86 million years for marsupial orders. With rate adjustments, the range of divergence estimates decreases to 53 to 133 million years for placentals versus 40 to 79 million years for marsupials. The effect of rate adjustments is most noticeable for carnivores and perissodactyls, where rates are slow, and proboscideans, where rates are fast. In agreement with studies based on nuclear genes, both unadjusted and rate-adjusted estimates of sequence divergence indicate that the majority of placental orders originated before the terminal Cretaceous extinction. Exceptions include the perissodactyl–carnivore split and cladogenesis among paenungulate orders. Most marsupial orders, in turn, may have originated in the early Tertiary although didelphimorphs, at least, appear to have split from other lineages in the late Cretaceous. Marsupial divergence times based on 12S rRNA data are in good agreement with estimates based on single-copy DNA hybridization and disagree with the suggestion of Hershkovitz (1992) that Dromiciops separated from other marsupials in the Jurassic.     

Cardiovascular effects of peptides related to the enkephalins and β-casomorphin

In the urethane-anesthetized rat, intravenous injections of morphine produced a short-lasting fall in heart rate and blood pressure. The fall in heart rate (which is vagal in origin) was used here to bioassay peptides related to the enkephalins [-enk] and β-casomorphin [β-C, H-Tyr-Pro-Phe-Pro-Gly-Pro-Ile-OH]. A > 10% decrease in heart rate was used as a quantal index of response and the intravenous ED50 [μmol/kg] were estimated as: methionine-enk, 1.3 ± .24; leucine-enk, 1.5 ± .20; [D-Ala², D-Leu⁵]-enk, 0.45 ± .05; [D-Ala², NMePhe⁴, Met(0)⁵-ol]-enk, 0.0011 ± .0002; H-Tyr-Arg-OH, > 2.0; β-C (1–7), > 2.0; β-C(1–5), > 2.0; β-C(1–4), > 4.0; β-C(1–3), > 2.0; morphine sulfate, 0.11 ± .03; and human β-endorphin,, 0.07 ± .01. One β-C derivative, H-Tyr-Pro-Phe-Pro-NH₂ [β-C(1–4)-NH₂], was active at 0.32 ± .08. Naloxone pretreatment blocked the bradycardia produced by the enkephalins and β-C(1–4)-NH₂. The bioassay described here, based on heart rate, may prove to be useful for the rapid detection and estimation of the $\text{in vivo}$ pharmacological activities of new opioid peptides.     

Effect of theβ-d-galactoside-binding lectin on cell to substratum and cell to cell adhesion of cells from the extraembryonic endoderm of the early chick blastoderm

Summary Cells from the extraembryonic endoderm of the gastrulating chick embryo contain a -d-galactoside-binding lectin inhibited by thiodigalactoside (TDG). When cell suspensions are cultured in stationary culture in the presence of exogenously added purified blastoderm lectin or TDG, their attachment to the substratum is delayed and decreased compared to controls. The cells take on a fibroblastic-like morphology and cell to cell contact becomes limited to localized areas of the cell surface. Many lectin or TDG-treated cells appear to be migrating over the substratum. This is in contrast to control cultures where the cells appear epithelial in morphology and tend to maximize their areas of apposition. These data suggest that the endogenous lectin may have a role to play in cell to substratum and cell to cell adhesion.     

Unigenic and multigenicI region control of the immune responses of mice to the GAT10 and GLΦ-GLT terpolymers

Recombinant strains of mice with known alleles in theI region of theH-2 complex were used to map theH-2 linked immune response genes controlling responsiveness to random terpolymers GAT¹⁰ and GL. TheIr-GAT gene was mapped to either theIA orIB subregions. In contrast, data obtained in the GL-GLT system indicated multigenic control. The responsiveness of the B10.A(3R), B10.A(5R), and B10.S(9R) recombinants indicated that one immune response gene,IrGL-GLT _A, mapped to the right ofIB, i.e., in theIC subregion. The nonresponsiveness of the B10.A(1R), B10.A(2R), B10.M(17R), and AQR mice having responderIC ^d alleles butIA ^k-IB ^k nonresponder alleles and the positive response of a (C57BL/6 × A/J)F₁ hybrid derived from two nonresponder parental strains indicated the presence of a second gene inIA-IB subregions,Ir-GL-GLT _B. The interaction between these two genes, each present in a differentI subregion, controls the immune response.     

Approaches to the simultaneous inactivation of metallo- and serine-β-lactamases

A series of cephalosporin-derived reverse hydroxamates and oximes were prepared and evaluated as inhibitors of representative metallo- and serine-β-lactamases. The reverse hydroxamates showed submicromolar inhibition of the GIM-1 metallo-β-lactamase. With respect to interactions with the classes A, C, and D serine β-lactamases, as judged by their correspondingly low K_m values, the reverse hydroxamates were recognized in a manner similar to the non-hydroxylated N–H amide side chains of the natural substrates of these enzymes. This indicates that, with respect to recognition in the active site of the serine β-lactamases, the OC–NR–OH functionality can function as a structural isostere of the OC–NR–H group, with the N–O–H group presumably replacing the amide N–H group as a hydrogen bond donor to the appropriate backbone carbonyl oxygen of the protein. The reverse hydroxamates, however, displayed k_cat values up to three orders of magnitude lower than the natural substrates, thus indicating substantial slowing of the hydrolytic action of these serine β-lactamases. Although the degree of inactivation is not yet enough to be clinically useful, these initial results are promising. The substitution of the amide N–H bond by N–OH may represent a useful strategy for the inhibition of other serine hydrolases.     

M?ssbauer spectroscopy applied to the oxidized and semi-reduced states of the iron-molybdenum cofactor of nitrogenase

M?ssbauer parameters at 125K for both the oxidized and semi-reduced states of FeMoco isolated from the MoFe protein of Azotobacter vinelandii nitrogenase of delta/Fe = 0.32 and 0.37 mm/s and delta Eq = 0.84 and 0.71 mm/s, respectively, are reported. FeMoco(ox) fits the Debye model perfectly from 4.2-125K and has a S = 0 ground state. FeMoco(ox) apparently contains 10-20% FeMoco(s-r) and vice versa, possibly as a result of the spontaneous oxidation phenomenon. Quantitation of the spectra indicates a Fe:Mo ratio of 5 +/- 1:1 and the similar quadrupole splittings and isomer shifts suggest a similar environment for all iron atoms.     

Crystallographic studies of the binding of ligands to the dicarboxylate site of Complex II,and the identity of the ligand in the “oxaloacetate-inhibited” state

Mitochondrial Complex II (succinate:ubiquinone oxidoreductase) is purified in a partially inactivated state, which can be activated by removal of tightly bound oxaloacetate (E.B. Kearney, et al., Biochem. Biophys. Res. Commun. 49 1115–1121). We crystallized Complex II in the presence of oxaloacetate or with the endogenous inhibitor bound. The structure showed a ligand essentially identical to the “malate-like intermediate” found in Shewanella Flavocytochrome c crystallized with fumarate (P. Taylor, et al., Nat. Struct. Biol. 6 1108–1112) Crystallization of Complex II in the presence of excess fumarate also gave the malate-like intermediate or a mixture of that and fumarate at the active site. In order to more conveniently monitor the occupation state of the dicarboxylate site, we are developing a library of UV/Vis spectral effects induced by binding different ligands to the site. Treatment with fumarate results in rapid development of the fumarate difference spectrum and then a very slow conversion into a species spectrally similar to the OAA-liganded complex. Complex II is known to be capable of oxidizing malate to the enol form of oxaloacetate (Y.O. Belikova, et al., Biochim. Biophys. Acta 936 1–9). The observations above suggest it may also be capable of interconverting fumarate and malate. It may be useful for understanding the mechanism and regulation of the enzyme to identify the malate-like intermediate and its pathway of formation from oxaloacetate or fumarate.     

Use of Fat-Fed Rats to Study the Metabolic and Vascular Sequelae of Obesity and ��-Adrenergic Antagonism

Obesity-associated cardiovascular disease exerts profound human and monetary costs, creating a mounting need for cost-effective and relevant in vivo models of the complex metabolic and vascular interrelationships of obesity. Obesity is associated with endothelial dysfunction and inflammation. Free fatty acids (FFA), generated partly through β-adrenergic receptor-mediated lipolysis, may impair endothelium-dependent vasodilation (EDV) by proinflammatory mechanisms. β-Adrenergic antagonists protect against cardiovascular events by mechanisms not fully defined. We hypothesized that β antagonists may exert beneficial effects, in part, by inhibiting lipolysis and reducing FFA. Further, we sought to evaluate the fat-fed rat as an in vivo model of obesity-induced inflammation and EDV. Control and fat-fed rats were given vehicle or β antagonist for 28 d. Serum FFA were measured to determine the association to serum IL6, TNFα, and C-reactive protein and to femoral artery EDV. Compared with controls, fat-fed rats weighed more and had higher FFA, triglyceride, leptin, and insulin levels. Unexpectedly, in control and fat-fed rats, β antagonism increased FFA, yet inflammatory cytokines were reduced and EDV was preserved. Therefore, reduction of FFA is unlikely to be the mechanism by which β antagonists protect the endothelium. These results reflect the need for validation of ex vivo models of obesity-induced inflammation and endothelial dysfunction, concurrent with careful control of dietary fat composition and treatment duration.Abbreviations: CRP, C-reactive protein; EDV, endothelium-dependent vasodilation; FFA, free fatty acids; FTI, flow–time integral; L-NAME, N_ω-nitro-L-arginine methyl ester; MAP, mean arterial pressure; PKA, protein kinase AThe prevalence of overweight and obese adults in the United States has increased by almost 20% over the last 3 decades.³⁶ Similar upward trends have been observed in persons between 6 and 19 y of age.³⁷ The obesity epidemic extracts a monetary cost of more than $92 billion on medical care alone⁵⁶ and a profound human price in the form of increased disease³⁵ and higher death rates.⁵⁰Obese adults have a higher risk of morbidity and mortality due to cardiovascular disease.⁵⁷ In health, endothelial cells that line the luminal surface of blood vessels release mediators that facilitate the appropriate regulation of multiple processes, including vascular permeability, inflammation and cell adhesion, coagulation, maintenance of intercellular matrix, lipid metabolism, and vascular reactivity.^25,42 Dysregulation of these processes favors inflammation, coagulation, and vasoconstriction. Not surprisingly, endothelial dysfunction as measured by impairment of endothelium-dependent vasodilation (EDV) is an early and reliable predictor of cardiovascular events in humans.^43,47Obese persons have increased serum free fatty acids (FFA).¹ Obesity^14,53 and FFA¹³ are associated with increased circulating inflammatory markers, specifically IL6, TNFα, and C-reactive protein (CRP). In addition, both obesity³² and FFA^8,46 are associated with impaired EDV, and FFA exert direct adverse inflammatory effects on the endothelium.¹⁸Partly in response to stimulation of β-adrenergic receptors, FFA are the principle moiety secreted from adipocytes. β-Adrenergic antagonist drugs reduce morbidity and mortality in patients with coronary artery disease^9,26 and affect both the myocardium^2,38 and vasculature.^5,51,55 The mechanisms by which β-adrenergic antagonists exert protection remain unclear, but reduced generation of FFA might play a role.The first aim of the present study was to determine whether β antagonism lowers serum FFA in fat-fed rats and whether the magnitude and direction of change in FFA is correlated with circulating inflammatory markers and EDV. The β₁- and β₃-receptor subtypes predominantly mediate lipolysis in rodent adipose;^12,30 however, to minimize the potential for compensatory upregulation of unopposed receptors in this study, the β₁β₂ antagonist propranolol was combined with the β₃ antagonist SR59230A (Sigma-Aldrich, St Louis, MO) to exert antagonism at all 3 receptor subtypes. To test whether the high-fat dietary treatment was associated with a metabolic milieu consistent with obesity, serum triglycerides, leptin, glucose, and insulin concentrations were measured.An inexpensive, valid, and physiologically relevant in vivo system would be valuable for studying the pandemic of obesity³¹ and related endothelial dysfunction.¹⁷ We are unaware of studies of whether long-term high-fat feeding affects in vivo EDV in the rat, although a study validating the use of high-resolution ultrasonography to measure in vivo flow-mediated vasodilation in normal rats was published recently.¹⁷ The second aim of our study was to investigate the fat-fed rat as a model of human diet-induced endothelial dysfunction. To retain the complex metabolic-vascular interplay that occurs in the intact organism, we used in vivo measures of EDV to assess the integrated physiologic response. In humans, the dilator response of peripheral vessels is associated with coronary EDV response.⁴⁸ We studied the rat femoral artery, with the aim of demonstrating changes in vasodilator responses in this easily isolated peripheral vascular bed.