α-Neurotoxin binding to acetylcholine receptor: Localization of the full profile of the cobratoxin-binding regions on the α-chain ofTorpedo californica acetylcholine receptor by a comprehensive synthetic strategy

Eighteen consecutive uniform overlapping synthetic peptides that spanned the entire extracellular part (residues 1–210) of the α-chain ofTorpedo californica acetylcholine receptor were screened for binding activity of¹²⁵I-labeled cobratoxin. Five toxin-binding regions were localized within residues 1–10, 32–41, 100–115, 122–150, and 182–198. The five toxin-binding regions may be distinct sites or, alternatively, different faces in one or more sites.     

Solution structure of N-terminal segment of hepatitis B virus surface antigen Pre-S1

ＨｅｐａｔｉｔｉｓＢｖｉｒｕｓ(ＨＢＶ),ｔｈｅｍａｊｏｒｃａｕｓａｔｉｖｅａｇｅｎｔｏｆｃｈｒｏｎｉｃｖｉｒａｌｈｅｐａｔｉｔｉｓａｎｄｌｉｖｅｒｃｉｒｒｈｏｓｉｓ,ｉｓｓｔｒｏｎｇｌｙｃｏｎｎｅｃｔｅｄｗｉｔｈｔｈｅｄｅｖｅｌｏｐｍｅｎｔｏｆｈｅｐａｔｏｃｅｌｌｕｌａｒｃａｒｃｉｎｏｍａ.ＯｎｅｏｆｔｈｅｍｏｓｔｒｅｍａｒｋａｂｌｅｆｅａｔｕｒｅｓｏｆＨＢＶｉｎｆｅｃｔｉｏｎｉｓｔｈａｔｉｎｆｅｃｔｅｄｃｅｌｌｓｐｒｏｄｕｃｅｍｕｌｔｉｐｌｅｔｙｐｅｓｏｆｖｉｒｕｓｒｅｌａｔｅｄｐａｒｔｉｃｌｅ…     

Microdistribution of sulfate-reducing bacteria in sediments of a hypertrophic lake and their response to the addition of organic matter

To clarify the ecological significance of the association of sulfate-reducing bacteria (SRB) with sediment particle size, SRB utilizing lactate (l-SRB), propionate (p-SRB) and acetate (a-SRB) were examined with different sizes of sediment particles in a hypertrophic freshwater lake using the anaerobic plate count method. The numbers ofl-SRB anda-SRB were 10⁴–10⁵ colony forming units (CFU) per ml in the 0–3 cm layer and 10²–10³ CFU ml⁻¹ in the 10–13 cm layer while the numbers ofp-SRB were one or two orders lower than those ofl-SRB anda-SRB. A sediment suspension was fractionated into four fractions (<1, 1–10, 10–94 and >94 μm). The highest proportions ofl-SRB anda-SRB were found in the 10–94 μm fraction: 66–97% forl-SRB and 53–98% fora-SRB. The highest proportion ofp-SRB was found in the >94 μm fraction (70–74%). These results indicate that most SRB were associated with sediment particles. One isolate from an acetate-utilizing enrichment culture was similar toDesulfotomaculum acetoxidans, a spore-forming sulfate-reducing bacterium. When lactate and sulfate were added to sediment samples,l-SRB anda-SRB in the <10 μm-fraction grew more rapidly than those in whole sediment for the first 2 days. This result suggests that nutrients uptake by free-living and small particle-associated (<10 μm) SRB is higher than that by SRB associated with larger particles.     

Theoretical prediction of antigenic sites in the Β-subunits of human choriogonadotropin and luteinizing hormone

Using hydrophilicity and recognition values of amino acids, the antigenic sites of theΒ-subunits of human choriogonadotropin and luteinizing hormone were computed from their amino acid sequences. Six antigenic sites were calculated for human choriogonadotropinΒ-subunits: residues 3–8, 17–22, 59–65,100–106,110–116 and 134–139. For luteinizing hormoneΒ-chain three antigenic sites were calculated: residues 17–22,59–65, and 100–106; all these three sites of luteinizing hormoneΒ being identical to the corresponding sites in human choriogonadotropinΒ. There was no antigenic site in luteinizing hormone that was also not found in human choriogonadotropin. On the other hand, there were unique determinants in human choriogonadotropin that were not found in luteinizing hormone; these determinants were residues 3–8, 110–116 and 134–139     

cDNA sequence and tissues expression analysis of lipoprotein lipase from common carp (<Emphasis Type="Italic">Cyprinus carpio</Emphasis> Var. Jian)

A full-length cDNA coding lipoprotein lipase (LPL) was cloned from liver of adult common carp (Cyprinus carpio Var. Jian) by RT-PCR and rapid amplification of cDNA ends (RACE) approaches. The cDNA obtained was 2,411 bp long with a 1,524 bp open reading frame (ORF) encoding 507 amino acids. This amino acid sequence contains two structural regions: N-terminus (24–354 residues) and C-terminus (355–507 residues). Before N-terminus, 1–23 residues is signal peptide, 6–23 residues is transmembrance helix. At N-terminus, some conversed functional sites were found, including two N-linked glycosylation sites Asn⁴¹ and Asn⁸⁸; one catalytic triad Ser¹⁷⁴, Asp¹⁹⁸ and His²⁸³; one conserved heparin-binding site Arg³²¹ to Arg³²⁴ (RKNR); eight cysteines residues Cys⁶⁹ and Cys⁸², Cys²⁵⁸ and Cys²⁸¹, Cys³⁰⁶ and Cys³²⁵, Cys³¹⁷ and Cys³²⁰ which are involved in four disulfide bridges; one polypeptide “lid” that participates in substrate specificity. At C-terminus, Asn⁴⁰¹ is another N-linked glycosylation site, and Trp⁴³⁴ and Trp⁴³⁵ (WW) is lipid-binding site. The amino acid sequence has a high similarity, and shows similar structural features to LPL of other species. Tissue distribution of LPL mRNA in liver, head kidney, mesenteric adipose tissue, heart and white muscle of common carp was analyzed by semi-quantitative RT-PCR method using β-actin gene as internal control. The result showed that the expressions of LPL mRNA were detected in all examined tissues of common carp. The expression levels of LPL in the mesenteric adipose tissue was highest among these tissues, following in liver and head kidney, and the lowest expression was found in heart and white muscle.     

Effect of malic acid on the growth kinetics of<Emphasis Type="Italic"> Lactobacillus plantarum</Emphasis>

The fermentation kinetics of Lactobacillus plantarum were studied in a specially designed broth formulated from commercially available, dehydrated components (yeast extract, trypticase, ammonium sulfate) in batch and continuous culture. During batch growth in the absence of malic acid, the specific growth rate was 0.20 h^–1. Malic acid in the medium, at 2 mM or 10 mM, increased the specific growth rate of L. plantarum to 0.34 h^–1. An increase in the maximum cell yield due to malic acid also was observed. Malic acid in the medium (12 mM) reduced the non-growth-associated (maintenance energy) coefficient and increased the biomass yield in continuous culture, based on calculations from the Luedeking and Piret model. The biomass yield coefficient was estimated as 27.4 mg or 34.3 mg cells mmol^–1 hexose in the absence or presence of malic acid, respectively. The maintenance coefficient was estimated as 3.5 mmol or 1.5 mmol hexose mg^–1 cell h^–1 in the absence or presence of malic acid. These results clearly demonstrate the energy-sparing effect of malic acid on the growth- and non-growth-associated energy requirements for L. plantarum. The quantitative energy-sparing effect of malic acid on L. plantarum has heretofore not been reported, to our knowledge.     

Novel Nociceptin Analogues: Synthesis and Biological Activity

Syntheses are described of the nociceptin (1–13) amide [NC(1–13)-NH₂] and of several analogues in which either one or both the phenylalanine residues (positions 1 and 4), the arginine residues (positions 8 and 12) and the alanine residues (positions 7 and 11) have been replaced by N-benzyl-glycine, N-(3-guanidino-propyl)-glycine and β-alanine, respectively. The preparation is also described of NC(1–13)-NH₂ analogues in which either galactose or N-acetyl-galactosamine are β-O-glycosidically linked to Thr⁵ and/or to Ser¹⁰. Preliminary pharmacological experiments on mouse vas deferens preparations showed that Phe⁴, Thr⁵, Ala⁷ and Arg⁸ are crucial residues for OP₄ receptor activation. Manipulation of Phe¹ yielded peptides endowed with antagonist activity but [Nphe¹] NC(1–13)-NH₂ acted as an antagonist still possessing weak agonist activity. Introduction of the βAla residue either in position 7 or 11 of the [Nphe¹] NC(1–13)-NH₂ sequence, abolished any residual agonist activity and [Nphe¹, βAla⁷] NC(1–13)-NH₂ and [Nphe¹, βAla¹¹] NC(1–13)-NH₂ acted as competitive antagonists only. Modification of both Ala⁷ and Ala¹¹ abolished the antagonist activity of [Nphe¹]NC(1–13)-NH₂ probably by hindering receptor binding. Changes at positions 10 and 11 gave analogues still possessing agonist activity. [Ser(βGal)¹⁰] NC(1–13)-NH₂ displayed an activity comparable with that of NC(1–13)-NH₂, [Ser(βGalNAc)¹⁰] NC(1–13)-NH₂ and [βAla¹¹] NC(1–13)-NH₂ were five and 10 times less active, respectively.The α-amino acid residues are of the l-configuration. Standard abbreviations for amino acid derivatives and peptides are according to the suggestions of the IUPAC-IUB Commission on Biochemical Nomeclature (1984), Eur. J. Biochem. 138, 9–37. Abbreviations listed in the guide published in (2003), J. Peptide Sci. 9, 1–8 are used without explanation.     

Positioning of the Alzheimer Aβ(1–40) peptide in SDS micelles using NMR and paramagnetic probes

NMR spectroscopy combined with paramagnetic relaxation agents was used to study the positioning of the 40-residue Alzheimer Amyloid β-peptide Aβ(1–40) in SDS micelles. 5-Doxyl stearic acid incorporated into the micelle or Mn²⁺ ions in the aqueous solvent were used to determine the position of the peptide relative to the micelle geometry. In SDS solvent, the two α-helices induced in Aβ(1–40), comprising residues 15–24, and 29–35, respectively, are surrounded by flexible unstructured regions. NMR signals from these unstructured regions are strongly attenuated in the presence of Mn²⁺ showing that these regions are positioned mostly outside the micelle. The central helix (residues 15–24) is significantly affected by 5-doxyl stearic acid however somewhat less for residues 16, 20, 22 and 23. This α-helix therefore resides in the SDS headgroup region with the face with residues 16, 20, 22 and 23 directed away from the hydrophobic interior of the micelle. The C-terminal helix is protected both from 5-doxyl stearic acid and Mn²⁺, and should be buried in the hydrophobic interior of the micelle. The SDS micelles were characterized by diffusion and ¹⁵N-relaxation measurements. Comparison of experimentally determined translational diffusion coefficients for SDS and Aβ(1–40) show that the size of SDS micelle is not significantly changed by interaction with Aβ(1–40). Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Ethanol production from hydrolysed agricultural wastes using mixed culture of <Emphasis Type="Italic">Zymomonas mobilis</Emphasis> and <Emphasis Type="Italic">Candida tropicalis</Emphasis>

Acid, alkaline and enzymatic hydrolysis of agricultural crop wastes were compared for yields of total reducing sugars with the hydrolysates being evaluated for ethanol production using a mixed culture of Zymomonas mobilis and Candida tropicalis. Acid hydrolysis of fruit and vegetable residues gave 49–84 g reducing sugars l⁻¹ and 29–32 g ethanol l⁻¹ was then obtained. Alkaline hydrolysis did not give significant amount of reducing sugars. Enzymatic hydrolysis of fruit and vegetable residues yielded 36–123 g reducing sugars l⁻¹ and 11–54 g ethanol l⁻¹.     

Correlations between foliar δ15N and nitrogen concentrations may indicate plant-mycorrhizal interactions

Nitrogen isotope measurements may provide insights into changing interactions among plants, mycorrhizal fungi, and soil processes across environmental gradients. Here, we report changes in δ¹⁵N signatures due to shifts in species composition and nitrogen (N) dynamics. These changes were assessed by measuring fine root biomass, net N mineralization, and N concentrations and δ¹⁵N of foliage, fine roots, soil, and mineral N across six sites representing different post-deglaciation ages at Glacier Bay, Alaska. Foliar δ¹⁵N varied widely, between 0 and –2‰ for nitrogen-fixing species, between 0 and –7‰ for deciduous non-fixing species, and between 0 and –11‰ for coniferous species. Relatively constant δ¹⁵N values for ammonium and generally low levels of soil nitrate suggested that differences in ammonium or nitrate use were not important influences on plant δ¹⁵N differences among species at individual sites. In fact, the largest variation among plant δ¹⁵N values were observed at the youngest and oldest sites, where soil nitrate concentrations were low. Low mineral N concentrations and low N mineralization at these sites indicated low N availability. The most plausible mechanism to explain low δ¹⁵N values in plant foliage was a large isotopic fractionation during transfer of nitrogen from mycorrhizal fungi to plants. Except for N-fixing plants, the foliar δ¹⁵N signatures of individual species were generally lower at sites of low N availability, suggesting either an increased fraction of N obtained from mycorrhizal uptake (f), or a reduced proportion of mycorrhizal N transferred to vegetation (T _r). Foliar and fine root nitrogen concentrations were also lower at these sites. Foliar N concentrations were significantly correlated with δ¹⁵N in foliage of Populus, Salix, Picea, and Tsuga heterophylla, and also in fine roots. The correlation between δ¹⁵N and N concentration may reflect strong underlying relationships among N availability, the relative allocation of carbon to mycorrhizal fungi, and shifts in either f or T _r. Received: 14 December 1998 / Accepted: 16 August 1999