Hb Natal or α2(minus Tyr-Arg)β2: A high oxygen affinity α chain variant with a deleted carboxy-terminus resulting from a TAC → TAA (Tyr → terminating codon) mutation in codon α140

The discovery is reported of a fast-moving α chain variant (Hb Natal) which is characterized by a shortened α polypeptide chain because of the deletion of the Tyr-Arg carboxy-terminal residues. Through amplification of appropriate segments of DNA and hybridization with synthetic oligonucleotide probes, it was possible to detect a C → A mutation in codon 140 of the α₂ globin gene, which causes a change in the codon for tyrosine to a terminating codon. Hb Natal or α₂(minus Tyr-Arg)β₂ has a high affinity for oxygen without a Bohr effect and heme-heme interaction. These results provide direct evidence for the importance of the tyrosine residue at α140 in the oxygenation-deoxygenation process.     

Synthetic Studies on Nephritogenic Glycosides. Synthesis of β-Glc-(1→6)-α-Glc-(l→6)-α-Glc-(1→Asn)

Gibberellins (GAs) A₉, A₁₅, A₁₉, A₂₀, A₂₉, A₃₅, A₄₄, A₅₀ and A₆₁ were identified by capillary gas chromatography/selected ion monitoring (GC/SIM) in immature seeds of loquat (Eriobotrya japonica Lindl). Furthermore, five unknown GA-like compounds with apparent parent ions of m/z 418, 504 or 506 (as methyl ester trimethylsilyl ether derivatives) were found by GC/mass spectrometry (GC/MS) in the biologically active fractions. The m/z 418 and 504 compounds may have been C-11β hydroxylated GA₉ and dehydro-GA₃₅, respectively. The bioassay and GC/MS results suggest that the major GAs were GA₅₀ and the five unknown GA-like compounds. In the immature seeds, at least two GA metabolic pathways may thus exist, one being the non-hydroxylation pathway of GA₁₅→GA₂₄→GA₉, and the other, the early C-13 hydroxylation pathway of GA₄₄→GA₁₉→GA₂₀→GA₂₉. A late C-11β hydroxylation pathway is also possible.     

Solution Properties of Antitumor Sulfated Derivative of α-(1→3)-D-Glucan from Ganoderma lucidum

Four fractions of a water-insoluble α-(1→3)-D-glucan GL extracted from fruiting bodies of Ganoderma lucidum were dissolved in 0.25 M LiCl/DMSO, and then reacted with sulfur trioxide-pyridine complex at 80°C to synthesize a series of water-soluble sulfated derivatives S-GL. The degree of substitution of DS was measured by using IR infrared spectra, elemental analysis, and ¹³C NMR to be 1.2-1.6 in the non-selective sulfation. Weight-average molecular weight M_w and intrinsic viscosity [η] of the sulfated derivatives S-GL were measured by multi-angle laser light scattering and viscometry. The M_w value (2.4×10⁴) of sulfated glucan S-GL-1 was much lower than that (44.5×10⁴) of original α-(1→3)-D-glucan GL-1. The Mark-Houwink equation and average value of characteristic ratio C_∞ for the S-GL in 0.2 M NaCl aqueous solution at 25°C were found to be: [η]=1.32×10^-3M_w^1.06 (cm³ g^-1) and 16, respectively, in the M_w range from 1.1×10⁴ to 2.4×10⁴. It indicated that the sulfated derivatives of the α-(1→3)-D-glucan in the aqueous solution behave as an expanded chain, owing to intramolecular hydrogen bonding or interaction between charge groups. Interestingly, two sulfated derivatives synthesized from the α-(1→3)-D-glucan and curdlan, a β-(1→3)-D-glucan, all had significant higher antitumor activity against Ehrlich ascites carcinoma (EAC) than the originals. The effect of expanded chains of the sulfated glucan in the aqueous solution on the improvement of the antitumor activity could not be negligible.     

Cystine-knot peptides engineered with specificities for α(IIb)β(3) or α(IIb)β(3) and α(v)β(3) integrins are potent inhibitors of platelet aggregation

A truncated form of the Agouti‐related protein (AgRP), a member of the cystine‐knot family, has shown promise as a scaffold for engineering novel peptides with new molecular recognition properties. In this study, we replaced a constrained six amino acid loop in AgRP with a nine amino acid loop containing an Arg–Gly–Asp integrin recognition motif, and randomized the neighboring residues to create a library of ～20 million AgRP variants. We displayed the AgRP mutants as fusions on the surface of yeast and used high‐throughput fluorescence‐activated cell sorting (FACS) to isolate peptides that bound specifically to the platelet integrin α_IIbβ₃, a clinically important target for the prevention and treatment of thrombosis. These AgRP peptides had equilibrium dissociation (K_D) constants for α_IIbβ₃ integrin ranging from 60 to 90 nM, and did not bind to α_vβ₃, α_vβ₅, or α₅β₁ integrins. Using an alternate library screening strategy, we identified AgRP peptides that bound to both α_IIbβ₃ and α_vβ₃ integrins with K_D values ranging from 40 to 70 nM and 20 to 30 nM, respectively, and did not bind to α_vβ₅ or α₅β₁ integrins. Unique consensus sequences were identified within both series of AgRP peptides suggesting alternative molecular recognition events that dictate different integrin binding specificities. In addition, the engineered AgRP peptides prevented platelet aggregation as well as or slightly better than the FDA‐approved cyclic peptide eptifibatide. Collectively, these data demonstrate that cystine‐knot peptides can be generated with high affinity and specificity to closely‐related integrins, and provide insights into molecular interactions between small, structured peptide ligands and their receptors. Copyright © 2010 John Wiley & Sons, Ltd.     

MMC and LD simulations of α-D-Glcp-(1→2)-α-D-Glcp-(1→3)-α-D-Glcp-OMe. A model for the terminal trisaccharide in glycoprotein precursors

The conformational flexibility and the dynamics of -D-Glcp-(12)--D-Glcp-(13)--D-Glcp-OMe (I) has been investigated by Metropolis-Monte Carlo with the HSEA (Hard Sphere Exo-Anomeric) force field and Langevin dynamics simulations employing two different CHARMm (Chemistry at HARvard Molecular Mechanics) force fields, CHEAT95 and PARM22. The conformational space spanned by the molecule is similar for the two former force fields but differ significantly for the latter. Hydrogen bonding between O2 and O4 of the title compound is analysed in comparison to NMR and preliminary results from X-ray powder diffraction studies. © 1998 Rapid Science Ltd     

Conformational analysis of α-d-Fuc-(1→4)-β-d-GlcNAc-OMe. One-dimensional transient NOE experiments and metropolis Monte Carlo simulations

Summary One-dimensional transient NOE build-up curves were measured for the synthetic disaccharide -d-Fuc-(14)--d-GlcNAc 1 utilizing Gaussian shaped pulses. Simulated build-up curves from Metropolis Monte Carlo simulations were compared to the experimental data. Disaccharide 1 is structurally related to methyl -d-maltoside in that it also contains an -(14) linkage, and it has the same configuration of groups around the glycosidic linkage. Analysis of NOEs in methyl -d-maltoside is restricted to those observed upon selective excitation of H1 because of severe spectral overlap. The situation is different in 1 where ¹H-NMR signals are well separated. Several interglycosidic NOEs were observed. The corresponding build-up curves allowed an accurate determination of the conformational preferences at the glycosidic linkage in 1. Comparison of experimental and theoretical NOE build-up curves showed clearly that rigid minimum-energy models cannot account for the experimental data. The best fit of experimental NOE build-up curves was obtained with theoretical curves from a 2×10⁶ step Metropolis Monte Carlo simulation with the temperature parameter set at 1000 K. Finally, it was observed that only the interglycosidic NOE H5/H6-pro-S significantly depends upon varying conformation distributions at the -(14)-glycosidic linkage, induced by choosing different temperature parameters for the Metropolis Monte Carlo simulations.     

The linkage of Hb Valletta [α2β287(F3)Thr→Pro] and Hb F-Malta-I [α2 Gγ2117(G19)His→Arg] in the Maltese population

Summary We have identified a new stable abnormal hemoglobin called Hb Valletta, which is characterized by a ThrPro substitution at position 87 of the chain. This mutation was found to be linked to that of the chain variant Hb F-Malta-I with a HisArg mutation at position 117 of the ^G chain. Both variants were detected in the blood samples of 34 Maltese and two Italian new-born babies with isoelectrofocusing and reversed phase high performance liquid chromatography. Similar analyses of cord blood from 388 additional Maltese newborns failed to identify either one of these two variants. Additional analyses of 353 Maltese adults (including 39 -thalassemia heterozygotes) resulted in the detection of two adult Hb Valletta heterozygotes. Dot-blot hybridization analyses of amplified DNA with a probe specific for the ^G-F-Malta-I variant showed that both also carried that mutation. These results show close linkage of the mutant forms of the ^G- and -globin genes, 27–28 kb apart, and a failure to identify chromosomes with either the Hb F-Malta-I mutation alone or with the Hb Valletta mutation alone, indicating a low recombination frequency.     

Studies on the stereoselective synthesis of a protected α-D-Gal-(1→2)-D-Glc fragment

A novel 1,2-cis stereoselective synthesis of protected α-D-Gal-(1→2)-D-Glc fragments was developed. Methyl 2-O-acetyl-3-O-allyl-4,6-O-benzylidene-α-D-galactopyranosyl-(1→2)-3-O-benzoyl-4,6-O-benzylidene-α-D-glucopyranoside (13), methyl 2-O-acetyl-3-O-allyl-4,6-O-benzylidene-α-D-galactopyranosyl-(1→2)-3,4,6-tri-O-benzoyl-α-D-glucopyranoside (15), methyl 2-O-acetyl-3-O-allyl-4,6-O-benzylidene-α-D-galactopyranosyl-(1→2)-3-O-benzoyl-4,6-O-benzylidene-β-D-glucopyranoside (17), and methyl 2-O-acetyl-3-O-allyl-4,6-O-benzylidene-α-D-galactopyranosyl-(1→2)-3,4,6-tri-O-benzoyl-β-D-glucopyranoside (19) were favorably obtained by coupling a new donor, isopropyl 2-O-acetyl-3-O-allyl-4,6-O-benzylidene-1-thio-β-D-galactopyranoside (2), with acceptors, methyl 3-O-benzoyl-4,6-O-benzylidene-α-D-glucopyranoside (4), methyl 3,4,6-tri-O-benzoyl-α-D-glucopyranoside (5), methyl 3-O-benzoyl-4,6-O-benzylidene-β-D-glucopyranoside (8), and methyl 3,4,6-tri-O-benzoyl-β-D-glucopyranoside (12), respectively. By virtue of the concerted 1,2-cis α-directing action induced by the 3-O-allyl and 4,6-O-benzylidene groups in donor 2 with a C-2 acetyl group capable of neighboring-group participation, the couplings were achieved with a high degree of α selectivity. In particular, higher α/β stereoselective galactosylation (5.0:1.0) was noted in the case of the coupling of donor 2 with acceptor 12 having a β-CH(3) at C-1 and benzoyl groups at C-4 and C-6.     

Kinetics of dextran-independent α-(1→3)-glucan synthesis by Streptococcus sobrinus glucosyltransferase I

Glucosyltransferase (GTF)-I from cariogenic Streptococcus sobrinus elongates the α-(1→3)-linked glucose polymer branches on the primer dextran bound to the C-terminal glucan-binding domain. We investigated the GTF-I-catalyzed glucan synthesis reaction in the absence of the primer dextran. The time course of saccharide production during dextran-independent glucan synthesis from sucrose was analyzed. Fructose and glucose were first produced by the sucrose hydrolysis. Leucrose was subsequently produced, followed by insoluble glucan [α-(1→3)-linked glucose polymers] after a lag phase. High levels of intermediate nigerooligosaccharide series accumulation were characteristically not observed during the lag phase. The results from the enzymatic activity of the acceptor reaction for the nigerooligosaccharide with a degree of polymerization of 2-6 and methyl α-D-glucopyranoside as a glucose analog indicate that the activity increased with an increase in the degree of polymerization. The production of insoluble glucan was numerically simulated using the fourth-order Runge-Kutta method with the kinetic parameters estimated from the enzyme assay. The simulated time course provided a profile similar to that of experimental data. These results define the relationship between the kinetic properties of GTF-I and the time course of saccharide production. These results are discussed with respect to a mechanism that underlies efficient glucan synthesis.     

The partial acid hydrolysis of a highly dextrorotatory fragment of the cell wall of Aspergillus niger. Isolation of the α-(1→3)-linked dextrin series

1. The cell-wall composition of Aspergillus niger has been investigated. Analysis shows the presence of six sugars, glucose, galactose, mannose, arabinose, glucosamine and galactosamine, all in the d-configuration, except that a small amount of l-galactose may be present. Sixteen common amino acids are also present. 2. The wall consists chiefly of neutral carbohydrate (73-83%) and hexosamine (9-13%), with smaller amounts of lipid (2-7%), protein (0.5-2.5%) and phosphorus (less than 0.1%). The acetyl content (3.0-3.4%) corresponds to 1.0mole/mole of hexosamine nitrogen. 3. A fractionation of the cell-wall complex was achieved, with or without a preliminary phenol extraction, by using n-sodium hydroxide. Though this caused some degradation, 30-60% of the wall could be solubilized (depending on the preparation). Analyses on several fractions suggest that fractionation procedures bring about some separation of components although not in a clear-cut fashion. 4. Cell-wall preparations were shown to yield a fraction having [alpha](D) approx. +240 degrees (in n-sodium hydroxide) and consisting largely of glucose. This was separated into two subfractions, one of which had [alpha](D)+281 degrees (in n-sodium hydroxide) and had properties resembling the polysaccharide nigeran; the other had [alpha](D) +231 degrees (in n-sodium hydroxide). It is suggested that nigeran is a cell-wall component.     

Organization of α-chain genes among Hb G-Philadelphia heterozygotes in association with Hb S, β-thalassemia,and α-thalassemia-2

The percentages of the -chain variant Hb G-Philadelphia (Hb G) or ₂ 68 AsnLys₂ were evaluated in 84 adult and 18 newborn heterozygotes. These included members of three families who were studied in more detail by nucleic acid hybridization techniques. The adult heterozygotes fell in two categories, one with a higher proportion of Hb G [46.5±1.0% (SD), N=21] and another with lower values (33.9±3.4%, N=63). Among the newborn heterozygotes, two babies fell in the category with the higher proportion of Hb G while 16 babies gave values between 25 and 34%. Studies of -chain gene organization on the parents of one neonate with a Hb G level of 27% at birth and 37% at 8 months excluded the presence of chromosomes with triplicated -chain genes which could lead to the ^0G/ genotype. Rather, these studies on five Hb G heterozygotes from three families confirmed the linkage between Hb G and a specific type of -thalassemia-2 associated with the presence of a 16-kbp Bgl II fragment which most probably carries the ^G locus since it has been found in 19 Hb G heterozygotes studied to date. The presence of an -thal-2 heterozygosity and three -chain genes (^0G/) was confirmed among Hb G heterozygotes with lower proportions of this variant. It is likely that the even lower values found in some newborn could arise through defective assembly of ^G- dimers. The presence of an -thal-2 homozygosity and two active -chain genes, one on each chromosome (^0G/⁰), was confirmed among heterozygotes with the higher proportion of Hb G. One of each of these categories was present in each of the three families investigated. This type of variability in the number of active -chain genes due to a heterozygosity or a homozygosity for -thalassemia-2 explains the trimodality of Hb S percentages among heterozygotes and the atypical hematological or biosynthetic features among patients with -thalassemia and sickle-cell syndromes.This research was supported by USPHS Research Grants HLB-05168 and HLB-15158 and by designated research funds of the Veterans Administration. This is Contribution No. 0693 of the Department of Cell and Molecular Biology, Medical College of Georgia, Augusta.     

Sequential synthesis and 13C-N.m.r. spectra of methyl β-glycosides of (1→4)-β-d-xylo-oligosaccharides

The reaction of 2,3-di-O-acetyl-4-O-benzyl-α,β-d-xylopyranosyl bromide (2) with methyl 2,3-di-O-acetyl-β-d-xylopyranoside gave methyl O-(2,3-di-O-acetyl-4-O-benzyl-β-d-xylopyranosyl)-(1→4)-2,3-di-O-acetyl-β-d-xylopyranoside (22). Catalytic hydrogenolysis of 22 exposed HO-4′ which was then condensed with 2. This sequence of reactions was repeated three more times to afford, after complete removal of protecting groups, a homologous series of methyl β-glycosides of (1→4)-β-d-xylo-oligosaccharides. ¹³C-N.m.r. spectra of the synthetic methyl β-glycosides (di- to hexa-saccharide) are presented together with data for six other, variously substituted, homologous series of (1→4)-d-xylo-oligosaccharides.     

Mapping Cod: Fisheries Science,Fish Harvesters’ Ecological Knowledge and Cod Migrations in the Northern Gulf of St. Lawrence

Some recent scholarship has focused on integrating local and/or traditional knowledge with conventional scientific information in fisheries management to improve the factual foundation of and strengthen support for management decisions. This article compares a sequence of historical and contemporary scientific texts and maps about the migrations and stock structure of cod in the Northern Gulf of St. Lawrence with texts and maps generated by the authors through the collection, aggregation and interpretation of commercial fish harvesters’ ecological knowledge. We find that the relationship between fisheries science and harvesters’ ecological knowledge is dynamic and has changed over time, and that both are ‘situated’ socially and ecologically. Overall, each paints an incomplete picture of cod movements and stock structure but the knowledge of harvesters provides a valuable complement to scientific information, particularly at the local scale, and has the potential to contribute to the identification of local cod stocks that are new to science and management. We end by considering how this case study informs the larger discussion about the challenges and potential benefits of the so-called integration project to bring together science and the ecological knowledge of fish harvesters.

Hb Costa Rica or · 2 ‚ 277(EF1)His→Arg: the first example of a somatic cell mutation in a globin gene

We have identified a minor hemoglobin component (∼5%) in the blood of a healthy Costa Rican female, but not in her mother and two brothers (father not studied), that has an His→Arg replacement at position β77 (Hb Costa Rica). No other amino acid replacements were observed and no β- or γ-chain-like peptides were present. Hb Costa Rica has a normal stability. Sequence analyses of numerous polymerase chain reaction (PCR)-amplified segments of DNA that contain exon 2 of the β gene failed to identify a CAC→CGC (His→Arg) mutation. The same was the case when cDNA was sequenced, indicating that a β-Costa Rica-mRNA could not be detected with this procedure. Gene mapping of genomic DNA with BglII, BamHI, and HindIII gave normal fragments only and with the same intensity as observed for the fragments of a normal control. The quantities of the β chain variants Hb J-Iran and Hb Fukuyama with related mutations at β77 vary between 30% and 45% in heterozygotes, whereas that of Hb F-Kennestone with the same His→Arg mutation but in the ^Gγ-globin gene, is a high 40%–45% (as percentage of total ^Gγ) in a heterozygous newborn. These different observations exclude a heterozygosity of the A→G mutation at codon β77, as well as a deletion comparable to that of Hbs Lepore or Kenya, or a β-globin gene duplication, and point to a nontraditional inheritance of Hb Costa Rica. Allele-specific amplification of cDNA with appropriate primers identified the presence of a low level of mutated mRNA in the reticulocytes of the patient, which was confirmed by dotblot analysis of the same material with ³²P-labeled probes. Comparable amplification products were not observed in genomic DNA. The A→G mutation apparently occurred in a somatic cell at a relatively early stage in the development of the hematopoietic cell system, and Hb Costa Rica accumulated through rapid cell divisions in patchy areas in the bone marrow (somatic mosaicism). An unequal distribution of Hb Costa Rica over the red cells supports this possibility. Received: 25 August 1995 / Revised: 13 December 1995