The Tryptophan Fluorescence of Tetracarbidium conophorum Agglutinin II and a Solution-Based Assay for the Binding of a Biantennary Glycopeptide

The plant lectin Tetracarbidium conophorum agglutinin II binds to glycoproteins and glycopeptides in a structurally specific manner [Animashaun et al., (1994) Glycoconjugate J. 11, 299–303]. We have characterized the steady-state and time-resolved fluorescence of the tryptophan residues of this lectin. The fluorescence (_ex = 295 nm, _em = 350 nm) decay is complex and can be described by four decay times with the following values: ₁ = 7.4nsec, ₁ = 0.22; ₂ = 2.9 nsec, ₂ = 0.25; ₃ = l.0 nsec, ₃ = 0.34; ₄ = 0.2 nsec, ₄ = 0.18. The addition of a biantennary glycopeptide to the lectin results in a quench and an 8 nm blue shift of the emission spectrum. The effect is saturable, and is described by an association constant of 1.8×10⁵ M^–1. The tryptophan fluorescence of Tetracarbidium conophorum agglutinin II may therefore be utilized to characterize thermodynamically the binding interactions between this lectin and complex glycoprotein.     

Studying Genetic Code by a Matrix Approach

Following Petoukhov and his collaborators, we use two length n zero-one sequences, α and β, to represent a length n genetic sequence ((a) || (b)){\alpha\choose\beta} so that the columns of ((a) || (b)){\alpha\choose\beta} have the following correspondence with the nucleotides: C ~ (0 || 0)C\sim{0\choose0} , U ~ (1 || 0)U\sim{1\choose0} , G ~ (1 || 1)G\sim{1\choose1} , A ~ (0 || 1)A\sim{0\choose1} . Using the Gray code ordering to arrange α and β, we build a 2 ⁿ ×2 ⁿ matrix C _n including all the 4 ⁿ length n genetic sequences. Furthermore, we use the Hamming distance of α and β to construct a 2 ⁿ ×2 ⁿ matrix D _n . We explore structures of these matrices, refine the results in earlier papers, and propose new directions for further research.     

Structural analysis of novel rhamnose-branched oligosaccharides from the glycophosphosphingolipids ofLeptomonas samueli

Mild alkaline hydrolysis of the glycophosphosphingolipids of the protozoanLeptomonas samueli liberated several phosphoinositol-containing oligosaccharides (PI-oligosaccharides), which were purified by high performance anion exchange chromatography. The oligosaccharides in the resulting four fractions were characterized by methylation analysis, fast atom bombardment mass spectrometry and two-dimensional nuclear magnetic resonance spectroscopy. The oligosaccharides contain the core structure Man(1–4)GlcN(1–6)-myo-inositol-1-OPO₃, and are substituted with 2mol of 2-aminoethylphosphonate per mol of oligosaccharide. The nonreducing ends of the oligosaccharides were terminated by rhamnose branched neutral and acidic xylose-containing penta-, hexa-, hepta- and octasaccharides, of which the three most abundant were shown to have the structures:

Genetic parameters and predicted selection responses for timber production traits in a Castanea sativa progeny trial: developing a breeding program

Total height, diameter, index volume, stem straightness, apical dominance, and survival were assessed at 8 years from seed in an open-pollinated progeny test of 36 families of European chestnut (Castanea sativa Miller) established at two sites in the Atlantic area of Galicia, Spain. Iterative spatial analysis was applied to eliminate the effect of the spatial dependence in the original data and to estimate accurately genetic parameters for evaluating the potential for selection of the measured trees. Spatial analysis was very beneficial for growth traits and survival, but less so if at all for form traits. Estimated individual heritabilities ranged from moderate to high for growth traits ([^(h)]_i² = 0.29 - 0.42 \widehat{h}_i^2 = 0.29 - 0.42 ) and stem straightness ([^(h)]_i² = 0.24 - 0.42 \widehat{h}_i^2 = 0.{24} - 0.{42} ). High coefficients of additive genetic variance were obtained for volume ( [^(\textC)]\textV_\textA = 36.5 - 41.5% \widehat{\text{C}}{{\text{V}}_{\text{A}}} = {36}.{5} - {41}.{5}\% ) and straightness ( [^(\textC)]\textV_\textA = 44.26 - 53.84% \widehat{\text{C}}{{\text{V}}_{\text{A}}} = {44}.{26} - {53}.{84}\% ). Phenotypic and estimated genetic correlations between growth traits were very high, and correlations between sites indicated that there was no important family × site interaction. No adverse correlations between traits were evident. The results indicate the ample potential for selection in the current progeny trial, where responses to within-family and combined selection for growth traits may be high. Accordingly, three selection scenarios were addressed with the aim to initiate the selection of individuals for implementing the Forest Breeding Plan of Galicia for European chestnut.     

HNCA-TOCSY-CANH experiments with alternate <Superscript>13</Superscript>C-<Superscript>12</Superscript>C labeling: a set of 3D experiment with unique supra-sequential information for mainchain resonance assignment

Described here is a set of three-dimensional (3D) NMR experiments that rely on CACA-TOCSY magnetization transfer via the weak ³ \textJ_{\textCa\textCa} ^{ 3} {\text{J}}_{{{\text{C}}\alpha {\text{C}}\alpha }} coupling. These pulse sequences, which resemble recently described ¹³C detected CACA-TOCSY (Takeuchi et al. 2010) experiments, are recorded in ¹H₂O, and use ¹H excitation and detection. These experiments require alternate ¹³C-¹²C labeling together with perdeuteration, which allows utilizing the small ³ \textJ_{\textCa\textCa} ^{ 3} {\text{J}}_{{{\text{C}}\alpha {\text{C}}\alpha }} scalar coupling that is otherwise masked by the stronger ¹J_CC couplings in uniformly ¹³C labeled samples. These new experiments provide a unique assignment ladder-mark that yields bidirectional supra-sequential information and can readily straddle proline residues. Unlike the conventional HNCA experiment, which contains only sequential information to the ^{1 3} \textC^a ^{ 1 3} {\text{C}}^{\alpha } of the preceding residue, the 3D hnCA-TOCSY-caNH experiment can yield sequential correlations to alpha carbons in positions i−1, i + 1 and i−2. Furthermore, the 3D hNca-TOCSY-caNH and Hnca-TOCSY-caNH experiments, which share the same magnetization pathway but use a different chemical shift encoding, directly couple the ¹⁵N-¹H spin pair of residue i to adjacent amide protons and nitrogens at positions i−2, i−1, i + 1 and i + 2, respectively. These new experimental features make protein backbone assignments more robust by reducing the degeneracy problem associated with the conventional 3D NMR experiments.     

New potent biphalin analogues containing p-fluoro-L-phenylalanine at the 4,4' positions and non-hydrazine linkers

We report the synthesis and the biological evaluation of two new analogues of the potent dimeric opioid peptide biphalin. The performed modification is based on the replacement of two key structural elements of the native biphalin, namely: the hydrazine bridge which joins the two palindromic moieties and the phenylalanine residues at the 4,4′ positions of the backbone. The new analogues 9 and 10 contain 1,2-phenylenediamine and piperazine, respectively, in place of the hydrazidic linker and p-fluoro-l-phenylalanine residues at 4 and 4′ positions. Binding values are: K_\texti^m = 0.51 \textnM K_{\text{i}}^{\mu } = 0.51\,{\text{nM}} and K_\texti^d = 12.8 \textnM K_{\text{i}}^{\delta } = 12.8\,{\text{nM}} for compound 9, K_\texti^m = 0.09 \textnM K_{\text{i}}^{\mu } = 0.09\,{\text{nM}} and K_\texti^d = 0.11 \textnM K_{\text{i}}^{\delta } = 0.11\,{\text{nM}} for analogue 10.     

The basic flow equations of electrophysiology in the presence of chemical reactions: I. Development of the equations

Starting from the basic flux equation, it is possible to obtain an integral form relating the current componentsI _i at an arbitrary pointr ₂ to the distribution of mobilities and concentrationsc _i, potential forces\(\bar \mu \), and chemical productivityp _i without any restrictive assumptions such as constant mobilities, constant field, steady state, or electrical neutrality. The equation is

$$\begin{gathered} I_i (r_2 ) = G_i (r_2 )\left[ {\Delta \bar \mu _i - \int_{r_1 }^{r_2 } {z_i } FA\left( {p_i - dc_i /dt} \right)\left( {\frac{1}{{G_i (r)}}} \right)dr} \right]; \hfill \\ G_i (r) = 1/\int_{r_1 }^r {\frac{{dr}}{{z_i^2 F^2 c_i u_i }}.} \hfill \\ \end{gathered} $$     

The Tryptophan Fluorescence of Tetracarbidium conophorum Agglutinin II and a Solution-Based Assay for the Binding of a Biantennary Glycopeptide

The plant lectin Tetracarbidium conophorum agglutinin II binds to glycoproteins and glycopeptides in a structurally specific manner [Animashaun et al., (1994) Glycoconjugate J. 11, 299–303]. We have characterized the steady-state and time-resolved fluorescence of the tryptophan residues of this lectin. The fluorescence (λ_ex = 295 nm, λ_em = 350 nm) decay is complex and can be described by four decay times with the following values: τ₁ = 7.4nsec, α₁ = 0.22; τ₂ = 2.9 nsec, α₂ = 0.25; τ₃ = l.0 nsec, α₃ = 0.34; τ₄ = 0.2 nsec, α₄ = 0.18. The addition of a biantennary glycopeptide $\begin{array}{*{20}c} {Gal\beta (1 \to 4)GlcNAc\beta (1 \to 2)Man\alpha (1 \to 6)\neg } \\ {Man\beta (1 \to 4)GlcNAC\beta (1 \to 4)GlcAc\beta (1 \to )\begin{array}{*{20}c} {Glu - Nh_2 } \\ | \\ {Asn} \\ | \\ {COOH} \\ \end{array} } \\ {Gal\beta (1 \to 4)GlcNAc\beta (1 \to 2)Man\alpha (1 \to 3)} \\ \end{array} $ to the lectin results in a quench and an 8 nm blue shift of the emission spectrum. The effect is saturable, and is described by an association constant of 1.8×10⁵ M^?1. The tryptophan fluorescence of Tetracarbidium conophorum agglutinin II may therefore be utilized to characterize thermodynamically the binding interactions between this lectin and complex glycoprotein.     

A directed alteration of ribonuclease specificity. Hydrolysis of polyribonucleotides containing modified cytosine bases

The action of ribonucleases on poly and oligoribonucleotides containing cytosine bases modified by methoxyamine and bisulphite was examined. Resistance of phosphodiester bonds in (Cp) _n Xp (where n 1 and X stands for A, G or U) to T₂ RNase hydrolysis was observed if substrates were modified chemically. The phenomenon formed the basis for isolation of (Cp) _n Xp blocks as an additional tool in sequence investigations. After modification of cytosine pancreatic RNase was unable to hydrolyse (Cp) _n Up blocks. Therefore the specificity of pyrimidyl RNase may be narrowed to uridyl RNase.Abbreviations cytidine modified with methoxyamine and bisulphite (5, 6-dihydro-6-sulpho-N⁴-methoxycytidine) - cytidine modified with methoxyamine (N⁴-methoxycytidine)     

Improved accuracy in measuring one-bond and two-bond <Superscript>15</Superscript>N,<Superscript>13</Superscript>C<Superscript>α</Superscript> coupling constants in proteins by double-inphase/antiphase (DIPAP) spectroscopy

An extension to HN(CO-α/β-N,C^α-J)-TROSY (Permi and Annila in J Biomol NMR 16:221–227, 2000) is proposed that permits the simultaneous determination of the four coupling constants ¹ J _{N′(i)Cα(i)}, ² J _HN(i)Cα(i), ² J _{Cα(i−1)N′(i)}, and ³ J _{Cα(i−1)HN(i)} in ¹⁵N,¹³C-labeled proteins. Contrasting the original scheme, in which two separate subspectra exhibit the ² J _CαN′ coupling as inphase and antiphase splitting (IPAP), we here record four subspectra that exhibit all combinations of inphase and antiphase splittings possible with respect to both ² J _CαN′ and ¹ J _N′Cα (DIPAP). Complementary sign patterns in the different spectrum constituents overdetermine the coupling constants which can thus be extracted at higher accuracy than is possible with the original experiment. Fully exploiting data redundance, simultaneous 2D lineshape fitting of the E.COSY multiplet tilts in all four subspectra provides all coupling constants at ultimate precision. Cross-correlation and differential-relaxation effects were taken into account in the evaluation procedure. By applying a four-point Fourier transform, the set of spectra is reversibly interconverted between DIPAP and spin-state representations. Methods are exemplified using proteins of various size.     

HA-detected experiments for the backbone assignment of intrinsically disordered proteins

We propose a new alpha proton detection based approach for the sequential assignment of natively unfolded proteins. The proposed protocol superimposes on following features: HA-detection (1) enables assignment of natively unfolded proteins at any pH, i.e., it is not sensitive to rapid chemical exchange undergoing in natively unfolded proteins even at moderately high pH. (2) It allows straightforward assignment of proline-rich polypeptides without additional proline-customized experiments. (3) It offers more streamlined and less ambiguous assignment based on solely intraresidual ¹⁵N(i)-¹³C′(i)-H^α(i) (or ¹⁵N(i)-¹³C^α(i)-H^α(i)) and sequential ¹⁵N(i + 1)-¹³C′(i)-H^α(i) (or ¹⁵N(i + 1)-¹³C^α(i)-H^α(i)) correlation experiments together with efficient use of chemical shifts of ¹⁵N and ¹³C′ nuclei, which show smaller dependence on residue type. We have tested the proposed protocol on two proteins, small globular 56-residue GB1, and highly disordered, proline-rich 47-residue fifth repeat of EspF_U. Using the proposed approach, we were able to assign 90% of ¹H^α, ¹³C^α, ¹³C′, ¹⁵N chemical shifts in EspF_U. We reckon that the HA-detection based strategy will be very useful in the assignment of natively unfolded proline-rich proteins or polypeptide chains.     

Alternate-site isotopic labeling of ribonucleotides for NMR studies of ribose conformational dynamics in RNA

Heteronuclear NMR spin relaxation studies of conformational dynamics are coming into increasing use to help understand the functions of ribozymes and other RNAs. Due to strong magnetic interactions within the ribose ring, however, these studies have thus far largely been limited to ¹³C and ¹⁵N resonances on the nucleotide base side chains. We report here the application of the alternate-site ¹³C isotopic labeling scheme, pioneered by LeMaster for relaxation studies of amino acid side chains, to nucleic acid systems. We have used different strains of E. coli to prepare mononucleotides containing ¹³C label in one of two patterns: Either C1′ or C2′ in addition to C4′, termed (1′/2′,4′) labeling, or nearly complete labeling at the C2′ and C4′ sites only, termed (2′,4′) labeling. These patterns provide isolated H spin systems on the labeled carbon atoms and thus allow spin relaxation studies without interference from scalar or dipolar coupling. Using relaxation studies of AMP dissolved in glycerol at varying temperature to produce systems with correlation times characteristic of different size RNAs, we demonstrate the removal of errors due to interaction in T ₁ measurements of larger nucleic acids and in T _1ρ measurements in RNA molecules. By extending the applicability of spin relaxation measurements to backbone ribose groups, this technology should greatly improve the flexibility and completeness of NMR analyses of conformational dynamics in RNA.     

The volumes of some compartment systems with sampling and loss from one compartment

If the concentrationc ₁(t)=∑ _i=1 ⁿ A _i exp (−α _i t) for one compartment, one presumes a linear catenaryn-compartment system without sinks and loss only from the same compartment, then the volumesV _i , rate constantsk _ij , and concentrationsc _i (t) in each compartment can be determined in terms of theA _i 's,A _i ′s, α _i ′s, the dose injectedD _o and the partition coefficientsr _ij =k _ij /k _ji . If the concentration would become uniform at equilibrium, then the total volume of distribution may be determined without knowledge ofr _ij or restriction to catenary configuration.     

A testcross procedure for selecting exotic strains to improve pure-line cultivars in predominantly self-fertilizing species

Methods for identifying germplasm carrying alleles with the potential to improve a particular single-cross hybrid have been proposed and discussed in recent years. There is a need for similar methods to be used in breeding crops for which pure-line cultivars, rather than hybrids, are the goal. The objective of this research was to develop a method to identify germplasm lines with the potential to contribute favorable alleles not present in a specified pure line or set of pure lines. Given a set of adapted pure lines (A ₁, A ₂ ..., A _m) to be improved and a set of germplasm lines (P ₁ P ₂ ..., P _f), the procedure consists of producing all f x m possible hybrids and evaluating them along with the parents. The testcross statistic T _ij is defined by T _ij=(F _ij–A _j)+(1–) (F _ij–P _i), where A _j, P _i, and F _ij represent the performance of thej ^th adapted line, the i ^th germplasm line, and their hybrid, respectively. The statistic is the mean value of T _ij over all adapted parents A _j. If =(1/2)(1+d), where d = the mean degree of dominance, then T _ij measures the potential for alleles from P _i to improve A _j and measures the potential for alleles from P _i to improve the set A ₁, A ₂ ..., A _m. Use of data on soybean and peanut hybrids published by other researchers suggests that the value assumed for d has little effect on the P _i chosen. The ability of the T _ij and statistics to identify germplasm strains carrying rare favorable alleles should be assessed in empirical studies.Joint contribution: OARDC (Journal Articale No. 161-94), USDAARS, Iowa Agriculture and Home Economics Expriment Station (Journal Paper No. J-16109; Project 2985), and Agreculture and Agri-Food Canada. Salaries and research support for S. K. St. Martin Provided by state and federal funds appropriated to the Ohio Agricultural Research and Development Center, Ohio State University     

Structure determination of the disialylated poly-(N-acetyllactosamine)-containingO-linked carbohydrate chains of equine chorionic gonadotropin

The disialylated poly-(N-acetyllactosamine)-containingO-linked oligosaccharide alditols, released by alkaline borohydride treatment of the enzymicallyN-deglycosylated β-subunit of equine chorionic chonadotropin, were purified by fast protein liquid chromatography (FPLC) on Mono Q and analysed by fast ion bombardment mass spectrometry (FAB-MS) and¹H-NMR spectroscopy. The identified oligosaccharide alditols have the following structure: $$\begin{gathered} Neu5Ac\alpha 2 - 3\left[ {Gal\beta 1 - 4GlcNAc\beta 1 - 3} \right]_{0 - 4} Gal\beta 1 - 4GlcNAc\beta 1 - 6 \hfill \\ \begin{array}{*{20}c} { \backslash } \\ { GalNAc - ol} \\ { /} \\ {Neu5Ac\alpha 2 - 3Gal\beta 1 - 3} \\ \end{array} \hfill \\ \end{gathered}$$      

On the biotopology of protein biosynthesis

The present paper is an attempt to outline a possible approach to the study of concrete cellular systems in terms of relational biology as developed by Rashevsky and Rosen. The basic ideas and the formalism of Rosen’s (M,R)-systems, proposed as a model of abstract biological systems, are used in order to represent the cellular protein biosynthesis. A diagram corresponding to the activation of amino acids and synthesis of amino-acyl-transfer RNA, the attachment of _t RNA to a specific codon of messenger RNA and peptide bond synthesis with the release of a protein molecule, is constructed. The systemM thus obtained for the synthesis of a proteinp _k receives a set of environmental inputs, that is, the twently naturally occurring amino acids and emits a single output, thep _k protein. The problem of noncontractibility of inputs in the system is then analyzed. In our context, it is found that the noncontractibility is not associated with the whole amino acid setS _pk but with an “essential amino acid set” , so that and represent the set of amino acids which can be replaced or absent. According to our considerations, the biochemical concept of “essential amino acid” acquires a new significance, that is, what seems “essential” is linked with the ability to form a giventRNA _t ∼a _i complex in a suitable augmented dependent set essential for the biosynthesis of a functional protein. Eventually the discussion of re-establishability leads to some important biological implications concerning the existence of ambiguous codons and the degeneracy phenomenon in the genetic code, as anecessary biochemical tool involved in adaptive processes.     

In Situ pH Measurement in Partly Frozen Aqueous Solution Using the Fluorescent Probe 8-Hydroxypyrene-1,3,6-Trisulfonic Acid

A method based on the fluorescence probe 8-hydroxypyrene-1,3,6-trisulfonic acid for in situ measurement of pH in partly frozen aqueous solutions was developed using multifrequency, phase-modulated fluorescence spectroscopy inherently correcting for light scattering. The probe was determined to have pK _a = 7.72 ± 0.03 at 25.0 °C extrapolated to zero ionic strength with as derived from temperature dependence (5 to 25 °C investigated). Ionic strength dependence of pK _a determined experimentally was described using Debye–Hückel formalism for ionic strength up to 3 M. Temperature and ionic strength dependence were combined to yield for determination of pH at subzero temperatures with α experimentally determined from the ratio between fluorescence intensity after excitation at 454 and 415 nm, α = FI(454 nm)/2.5·FI(415 nm). Fluorescence could be described as a decay of a single excited state with a fluorescence life time of 5.40 ± 0.05 ns at 25 °C, and excited state acid–base equilibration was shown not to interfere with the pH measurement. Using the method, pH of a 0.25 M phosphate buffer with pH = 6.8 at 25 °C was shown to decrease gradually to pH = 4.2 in the ice slurry at −13 °C.     

Studies in mathematical biosociology of imitative behavior: I. Effects of income distribution

In connection with previous studies (Rashevsky,Mathematical Biology of Social Behavior, chap. xii), a situation is investigated in which the two mutually exclusive possible behaviors of a society consist of the desire to keep the present socioeconomical situation and the desire to change it inany way. The psychophysiological tendency ϕ towards either of the behaviors is considered to be proportional to the difference between the actual incomei of the individual and his needsi′. Assuming that the distribution functionN ₁(i′) of the needs is a given characteristic of the population, it is shown that the distribution functionN(ϕ) of ϕ in the society can be derived fromN ₁(i′) and from the distributionN ₂(i) of the incomesi. A particular case is worked out as an example. Conditions of stability of a socioeconomic structure are studied in their dependence on the income distribution.     

Soil temperature and biotic factors drive the seasonal variation of soil respiration in a maize (Zea mays L.) agricultural ecosystem

The diurnal and seasonal variation of soil respiration (SR) and their driving environmental factors were studied in a maize ecosystem during the growing season 2005. The diurnal variation of SR showed asymmetric patterns, with the minimum occurring around early morning and the maximum around 13:00 h. SR fluctuated greatly during the growing season. The mean SR rate was 3.16 μmol CO₂ m⁻² s⁻¹, with a maximum of 4.87 μmol CO₂ m⁻² s⁻¹ on July 28 and a minimum of 1.32 μmol CO₂ m⁻² s⁻¹ on May 4. During the diurnal variation of SR, there was a significant exponential relationship between SR and soil temperature (T) at 10 cm depth: . At a seasonal scale, the coefficient α and β fluctuated because the biomass (B) increased α, and the net primary productivity (NPP) of maize markedly increased β of the exponential equation. Based on this, we developed the equation to estimate the magnitude of SR and to simulate its temporal variation during the growth season of maize. Most of the temporal variability (93%) in SR could be explained by the variations in soil temperature, biomass and NPP of maize. This model clearly demonstrated that soil temperature, biomass and NPP of maize combined to drive the seasonal variation of SR during the growing season. However, only taking into account the influence of soil temperature on SR, an exponential equation over- or underestimated the magnitude of SR and resulted in an erroneous representation of the seasonal variation in SR. Our results highlighted the importance of biotic factors for the estimation of SR during the growing season. It is suggested that the models of SR on agricultural sites should not only take into account the influence of soil temperature, but also incorporate biotic factors as they affect SR during the growing season.     

Modification of the data-processing method for the turbidimetric bioassay of nisin

The data processing method of the turbidimetric bioassay of nisin was modified to facilitate its industrial application. The influence of the initial indicator concentration was minimized by a redefined specific dose of the bacteriocin as the quotient between the titer of the added bacteriocin and the initial population density of the indicator in the suspension. It was found that d _c = 0.125 μg ml⁻¹ was the critical dose of nisin that can cause a complete inhibition of the indicator, Pediococcus acidilactici UL5, with an initial OD of 0.135. To eliminate the interference of the cell debris, an equation, , exploiting d _c, was formulated to obtain the intrinsic survival proportion. The use of the specific dose of the bacteriocin and the intrinsic survival proportion as parameters of the dose/response curve greatly enhanced its repeatability and feasibility. A dual-dosage approach was developed to further simplify the conventional standard dose/response curve method.