Isolation of Tryptic Peptides from the lie Chain of Ricin D and the Sequences of Some Tryptic Peptides Including N- and C-Terminal Peptides

Twenty tryptic peptides were isolated from the performic acid-oxidized He chain of ricin D by Dowex 1 × 2 column chromatography followed by paper chromatography. The amino acids contained in these peptides accounted for 218 out of 266 residues in the whole protein. The amino acid sequences of nine peptides were determined by manual liquid phase or automatic solid phase Edman degradation, and N- and C-terminal sequences of the He chain of ricin D were established to be NH₂–Ile–Phe–Pro–Lys–Gln–Tyr–Pro–Ile–Ile– and Cys–Ala–Pro–Pro–Pro–Ser–Ser–Gln–Phe, respectively.     

Ascidiacyclamides containing oxazoline and thiazole motifs assume square conformations and show high cytotoxicity

Four cyclic octapeptides were designed from ascidiacyclamide [cyclo(–Ile–Oxz–D ‐Val– Thz–)₂] (ASC, 1 ) to investigate the effects of oxazoline (Oxz) and thiazole (Thz) rings on the structures and cytotoxicities of the peptides. cyclo(–Ile–Thz–D ‐Val–Oxz–)₂ ( 2 ) had the same number of Oxz and Thz rings as ASC, but the ring positions were switched. cyclo(–Ile–Oxz–D ‐Val–Thz–Ile–Thz–D ‐Val–Thz–) ( 3 ) and cyclo(–Ile–Thz–D ‐Val–Oxz–Ile–Thz–D ‐Val–Thz–) ( 4 ) contained one Oxz and three Thz rings within the molecule. All Oxz rings were substituted with Thz in cyclo(–Ile–Thz–D ‐Val–Thz–)₂ ( 5 ). These analogues had new Oxz and Thz blocks forming the 24‐membered ring. Based on CD spectra and X‐ray diffraction analyses, the structures of all four analogues were classified as square ASC forms. But the structures of 2 and 5 differed from the original square form of 1 , and they showed no cytotoxicity. The structure of 3 was very similar to that of 1 , and 3 showed 10 times greater cytotoxicity than 1 . Although no definite structure of 4 was obtained, it showed three times greater cytotoxicity than 1 . It appears that the position and number of Oxz residues are essential determinants in the structure‐cytotoxicity relationship of ASC analogues.     

Peptides with 6-Aminohexanoic Acid: Synthesis and Evaluation as Plasmin Inhibitors

Fifteen new peptide derivatives of ?-aminocaproic acid (EACA) containing the known fragment –Ala–Phe–Lys– with an affinity for plasmin were synthesised in the present study. The synthesis was carried out a solid phase. The following compounds were synthesised: H–Phe–Lys–EACA–X, H–d-Ala–Phe–Lys–EACA–X, H–Ala–Phe–Lys–EACA–X, H–d-Ala–Phe–EACA–X and H–Ala–Phe–EACA–X, where X = OH, NH₂ and NH–(CH₂)₅–NH₂. All peptides, except for those containing the sequence H–Ala–Phe–EACA–X, displayed higher inhibitory activity against plasmin than EACA. The most active and selective inhibitor of plasmin was the compound H–d-Ala–Phe–Lys–EACA–NH₂ which inhibited the amidolytic activity of plasmin (IC₅₀ = 0.02 mM), with the antifibrinolytic activity weaker than EACA. The resulting peptides did not affect the viability of fibroblast cells, colon cancer cell line DLD-1, breast MCF-7 and MDA-MB-231 cell lines.     

The effect of temperature and salt concentration on the circular dichroism exhibited by unionized derivatives of L-alanine in aqueous solution

The circular dichroism of Ac–Ala–NHMe, cyclo(–Ala–Ala–), Ac–Ala–OMe, Ac–Ala–Ala–OMe, and Ac–Ala–Ala–Ala–OMe has been measured in water and in aqueous salt solutions as a function of temperature. Only cyclo(–Ala–Ala–) exhibits circular dichroism which is independent of temperature. Each of the linear derivatives of L -alanine exhibits a positive circular dichroism in the range 208–218 nm at 15°C in water. Heating reduces the intensity of the positive circular dichroism, and only Ac–Ala–OMe retains positive circular dichroism at 75°C in water. Isothermal addition of salts produces changes in the circular dichroism of linear derivatives of L -alanine which resemble those seen on heating. The relative effectiveness of the salts tested, at a concentration of 4M, is LiCl ? KCl = NaCl < MgCl₂ ? CaCl₂ ? NaClO₄. The circular dichroism of cyclo(–Ala–Ala–) is also affected by the salts. Extrapolation of the results obtained with Ac–Ala–OMe, Ac–Ala–Ala–OMe, and Ac–Ala–Ala–Ala–OMe to a long polypeptide with a –CH₂R side chain in the L -configuration leads to the conclusion that this polypeptide should exhibit a temperature-dependent salt-sensitive positive circular dichroism between 208 and 218 nm when it exists as a statstical coil.     

Calculation of the Three-Dimensional Structures of Two Antigenic Sequences, Pro–Pro–Pro–Pro–Asn–Pro–Asn–Asp–Pro and Pro–Pro–Pro–Pro–Asn–Pro–Asn–Asp–Pro–Pro–Pro, of the Circumsporozoite Protein From a Malaria-Causing Plasmodium

Using the chain build-up procedure based on the program ECEPP, we have computed the lowest energy structures for two terminally blocked subsequences from the antigenic circumsporozoite protein of Plasmodium berghei, that is known to cause malaria in animals. The full antigenic sequence is an octapeptide proline-rich tandem repeat, (Pro–Pro–Pro–Pro–Asn–Pro–Asn–Asp)₂. We computed the structures for the first octapeptide plus one Pro from the second octapeptide, terminally blocked CH₃CO–Pro–Pro–Pro–Pro–Asn–Pro–Asn–Asp–Pro–NHCH₃ as well as the first octpeptide with an additional three Pro residues from the adjoining unit, i.e., CH₃CO–Pro–Pro–Pro–Pro–Asn–Pro–Asn–Asp–Pro–Pro–Pro–NHCH₃. We find that the first sequence adopts a number of different low energy structures, the most probable of which has a probability of occurrence of 56 %. Addition of two more Pro residues results in the adoption a single, unique lowest energy structure that has a probability of occurrence of over 95 % without solvation effects and 86 % when solvation effects are included in the calculations. We predict that this structure may be the one recognized as a major antigenic determinant.     

VARIATION IN APPLE CYTOCHIMERAS

Diploid-tetraploid cytochimeras of apple were investigated by cytological examination of buds from branches selected by the characteristics of the fruits, flowers or pollen. Eight types of cytochimeras were identified on the basis of relative size of cells, nuclei and metaphase mitoses in the apical meristem (protomeristem) and mitoses in meristematic primary tissues. There appeared to be five apical layers which contributed to the stem and four which contributed to the leaves and flower parts. Buds of various cytochimeral patterns (designated by a formula giving the ploidy of the apical layers) were found on some bearing trees propagated from known cytochimeral sources. The most frequently associated types were (a) 2–4–2–2–2 and 2–4–4–4–4 (and 2–4–2–4–4 in one cultivar), or (b) 2–2–2–4–4 and 2x. Some sports were uniformly 2–4–4–4–4. The stability of apple cytochimeras under normal conditions appeared greater in some eultivars than in others. Sprouts from severely pruned 2–2–4–4–4 trees were more variable than unpruned branches. Buds of shoots which grew from radiation-damaged buds were more variable than those from non-irradiated buds and included types not yet found by branch selection. Cytochimeral variation was interpreted to be due to layer replacement resulting from infrequent periclinal divisions in apical or axillary meristems, or from wounding of meristems by ionizing radiation.     

ACE Inhibitory and Antioxidant Activities of Novel Peptides from <Emphasis Type="Italic">Scorpaena notata</Emphasis> By-product Protein Hydrolysate

This study describes the isolation of angiotensin I converting enzyme and antioxidative peptides from head protein hydrolysate of red scorpionfish (Scorpaena notata) prepared by treatment with a protease from the fungus Penicillium digitatum. After ultrafiltration, three peptides were isolated by a two-step procedure: size exclusion chromatography on a Toyopearl HW-40 followed by reversed-phase high performance liquid chromatography (RP-HPLC) with a high purification yield of 2.22 mg of peptide/g of initial protein. Active peptides were then identified by nanoscale liquid chromatography coupled to tandem mass spectrometry (nanoLC/MS–MS), corresponding to the following sequences: Gln–Gln–Pro–His–Ser–Arg–Ser–Lys–Gly–Phe–Pro–Gly–Pro (1424.724 Da), Gly–Gln–Lys–Ser–Val–Pro–Glu–Val–Arg (1000.565 Da) and Val–Glu–Gly–Lys–Ser–Pro–Asn–Val (830.448 Da). Peptides D-I, E-I and F-I showed high angiotensin-I converting enzyme inhibitory activity with an IC₅₀ values of 0.98, 1.69 and 1.44 µM, respectively as well as a synergistic antioxidant activity between the different fractions. Thus, we have demonstrated that underutilized wastes can be valorized by production of peptides that can be used as potential therapeutic compounds active against oxidative stress and hypertension.     

Mapping of the antibody-binding regions on botulinum neurotoxin H-chain domain 855–1296 with antitoxin antibodies from three host species

Botulism due to food poisoning is caused mainly by protein toxins, botulinum neurotoxins (BoNTs), produced byClostridium botluinum in seven known immunological serotypes. These are the most potent toxins and poisons known. BoNT effects blockade of neuromuscular transmission by preventing neurotransmitter release. Human botulism is most frequently caused by types A, B, and E. Recent studies have shown that immunization with a 43-kDa C-terminal fragment (H_C, residues 860–1296) of BoNT/A affords excellent protection against BoNT/A poisoning. We raised antibodies (Abs) against BoNT/A in horse, and against pentavalent toxoid (BoNTs A, B, C, D, E) in human volunteers and outbred mice. Thirty-one 19-residue peptides that started at residue 855, overlapped consecutively by 5 residues, and encompassed the entire length of the H_C of BoNT/A were synthesized and used for mapping the Ab-binding regions recognized by the anti-BoNT/A antisera. Horse Abs against BoBT/A were bound by peptides 855–873, 939–957, 1079–1097/1093–1111 overlap, 1191–1209/1205–1223 overlap, 1261–1279 and 1275–1296. In addition, peptides 883–901, 911–929, 995–1013, 1023–1041/1037–1055 overlap, 1121–1139, and 1149–1167 gave low, but significant and reproducible, binding. With human antisera, high amounts of Abs were bound by peptides 869–887, 925–943, 981–999, 995–1013, 1051–1069, and 1177–1195. In addition, lower amounts of Abs were bound by peptides 911–929, 939–957, 967–985, and the overlaps 1121–1139/1135–1153 and 1247–1265/1261–1279/1275–1296. With outbred mouse antisera, high amounts of Abs were bound by peptides 869–887, 1051–1069, and 1177–1195, while peptides 939–957, 995–1013, 1093–1111, and 1275–1296 bound lower amounts of Abs. The results indicate that horse antiserum against BoNT/A or human and mouse (outbred) antisera against the toxoid recognized similar regions on BoNT/A, but exhibited some boundary frame shifts and differences in immunodominance of these regions among the antisera. Selected synthetic epitopes will be used as immunogens to stimulate active or passive (by Ab transfer) immunity against toxin poisoning.     

Chemical structures of oligosaccharides in milks of the American black bear (Ursus americanus americanus) and cheetah (Acinonyx jubatus)

The milk oligosaccharides were studied for two species of the Carnivora: the American black bear (Ursus americanus, family Ursidae, Caniformia), and the cheetah, (Acinonyx jubatus, family Felidae, Feliformia). Lactose was the most dominant saccharide in cheetah milk, while this was a minor saccharide and milk oligosaccharides predominated over lactose in American black bear milk. The structures of 8 neutral saccharides from American black bear milk were found to be Gal(β1–4)Glc (lactose), Fuc(α1–2)Gal(β1–4)Glc (2′-fucosyllactose), Gal(α1–3)Gal(β1–4)Glc (isoglobotriose), Gal(α1–3)[Fuc(α1–2)]Gal(β1–4)Glc (B-tetrasaccharide), Gal(α1–3)[Fuc(α1–2)]Gal(β1–4)[Fuc(α1–3)]Glc (B-pentasaccharide), Fuc(α1–2)Gal(β1–4)[Fuc(α1–3)]GlcNAc(β1–3)Gal(β1–4)Glc (difucosyl lacto-N-neotetraose), Gal(α1–3)Gal(β1–4)[Fuc(α1–3)]GlcNAc(β1–3)Gal(β1–4)Glc (monogalactosyl monofucosyl lacto-N-neotetraose) and Gal(α1–3)Gal(β1–4)GlcNAc(β1–3)Gal(β1–4)Glc (Galili pentasaccharide). Structures of 5 acidic saccharides were also identified in black bear milk: Neu5Ac(α2–3)Gal(β1–4)Glc (3′-sialyllactose), Neu5Ac(α2–6)Gal(β1–4)GlcNAc(β1–3)[Fuc(α1–2)Gal(β1–4)GlcNAc(β1–6)]Gal(β1–4)Glc (monosialyl monofucosyl lacto-N-neohexaose), Neu5Ac(α2–6)Gal(β1–4)GlcNAc(β1–3)[Gal(α1–3)Gal(β1–4)GlcNAc(β1–6)]Gal(β1–4)Glc (monosialyl monogalactosyl lacto-N-neohexaose), Neu5Ac(α2–6)Gal(β1–4)GlcNAc(β1–3){Gal(α1–3)Gal(β1–4)[Fuc(α1–3)]GlcNAc(β1–6)}Gal(β1–4)Glc (monosialyl monogalactosyl monofucosyl lacto-N-neohexaose), and Neu5Ac(α2–6)Gal(β1–4)GlcNAc(β1–3){Gal(α1–3)[Fuc(α1–2)]Gal(β1–4)[Fuc(α1–3)]GlcNAc(β1–6)}Gal(β1–4)Glc (monosialyl monogalactosyl difucosyl lacto-N-neohexaose). A notable feature of some of these milk oligosaccharides is the presence of B-antigen (Gal(α1–3)[Fuc(α1–2)]Gal), α-Gal epitope (Gal(α1–3)Gal(β1–4)Glc(NAc)) and Lewis x (Gal(β1–4)[Fuc(α1–3)]GlcNAc) structures within oligosaccharides. By comparison to American black bear milk, cheetah milk had a much smaller array of oligosaccharides. Two cheetah milks contained Gal(α1–3)Gal(β1–4)Glc (isoglobotriose), while another cheetah milk did not, but contained Gal(β1–6)Gal(β1–4)Glc (6′-galactosyllactose) and Gal(β1–3)Gal(β1–4)Glc (3′-galactosyllactose). Two cheetah milks contained Gal(β1–4)GlcNAc(β1–3)[Gal(β1–4)GlcNAc(β1–6)]Gal(β1–4)Glc (lacto-N-neohexaose), and one cheetah milk contained Gal(β1–4)Glc-3’-O-sulfate. Neu5Ac(α2–8)Neu5Ac(α2–3)Gal(β1–4)Glc (disialyllactose) was the only sialyl oligosaccharide identified in cheetah milk. The heterogeneity of milk oligosaccharides was found between both species with respect of the presence/absence of B-antigen and Lewis x. The variety of milk oligosaccharides was much greater in the American black bear than in the cheetah. The ratio of milk oligosaccharides-to-lactose was lower in cheetah (1:1–1:2) than American black bear (21:1) which is likely a reflection of the requirement for a dietary supply of N-acetyl neuraminic acid (sialic acid), in altricial ursids compared to more precocial felids, given the role of these oligosaccharides in the synthesis of brain gangliosides and the polysialic chains on neural cell adhesion.

     

Replication and extension of association of choline acetyltransferase with nicotine dependence in European and African American smokers

Choline acetyltransferase is critical in the synthesis of acetylcholine and regulation of cholinergic neuron functions. We recently reported association of the encoding gene ChAT with both smoking cessation and nicotine dependence (ND) in two independent European American (EA) samples; however, in the replication sample, only limited SNPs partially covering the gene were examined. In this study, we examined the association of 14 SNPs, which cover the entire gene, with ND, assessed by smoking quantity (SQ), heaviness of smoking index (HSI), and Fagerström Test for ND (FTND), in 2,037 subjects from 602 families of African American (AA) or EA origin. Individual SNP-based association analysis revealed that five SNPs showed nominal association with at least one ND measure in one of the samples (P = 0.022–0.042); none remained significant after correction for multiple testing. Haplotype-based association analysis revealed that haplotypes G–G–A–C, formed by rs1880676–rs3810950–rs10082479–rs8178990 (P = 0.005–0.0178), and G–G–T–C–G–C, formed by rs1880676–rs3810950–rs10082479–rs8178990–rs3793790–rs12266458 (P = 0.00247–0.00468), displayed significant association with all three ND measures in the AA sample, as did haplotype T–C–G–A–T, formed by rs12266458–rs11101191–rs8178991–rs4838544–rs4838547 (P = 0.00741–0.0103), in the EA sample. All these detected haplotype-based associations remained significant after correction for all major haplotypes for a given SNP combination. Together, our findings, in conjunction with the previous report of the association, warrant further investigation of ChAT in ND.     

COLCHIPLOIDY AND HISTOLOGICAL IMBALANCE IN TRIPLOID APPLE AND PEAR

Colchicine treatment of the triploid apple varieties ‘Paragon’ and ‘Stayman’ resulted in 6–3–3 and 3–6–6 chimeras, and 6–3–3 chimera in ‘Baldwin.’ Treatment of a triploid pear selection ‘U.S. 44–3–1’ resulted in totally 6x as well as 6–3–3 and 3–6–6 chimeras. As triploid plants are not suitable for breeding, sexually hexaploid forms (totally 6x or chimeral 3–6–6) of high-quality triploid apples and pears may become very useful as breeding material. The primary purpose of treating the ‘Stayman’ apple was to obtain a 6–3–3 chimera to find out if large cells in the epidermis would reduce or eliminate the severe cracking of fruit of this variety which occurs in some seasons. Chimeral forms in the other plants were to serve as material for comparison with the ‘Stayman’ variety. Observations on the fruit-cracking problem will have to wait until the chimeral plants fruit. Hexaploid tissue in chimeral shoots of all affected plants has grown more slowly than triploid tissue, causing various degrees of malformation of the leaves. Similar reduction in growth due to restricted ploidization in 4–2–2 or 2–4–4 chimeras has not resulted in many other plants, including apples and pears, but was reported in an 8–4–4 chimera in cranberry. Slower growth of 6x tissue in the apple and pear may be attributed to the presence of multiple homologous genes in the duplicated chromosome forms of these particular triploid plants.     

Novel stable analogues of the neurotensin C-terminal hexapeptide containing unnatural amino acids

Amino Acids - Neurotensin (NT) (pGlu–Leu–Tyr–Glu–Asn–Lys–Pro–Arg–Arg–Pro–Tyr–Ile–Leu) exerts a dual function as a...     

The Status of the Hemostasis System under the Influence of Proline Peptides during the Development of Experimental Metabolic Syndrome

A comparative study of the influence of regulatory proline peptides Pro–Gly–Pro, Pro–Arg–Pro, Pro–Gly–Pro–Leu, and Arg–Pro–Gly–Pro on the state of the hemostasis system was carried out in an experiment on male rats with metabolic syndrome. Under these conditions, repeated (7-fold) intranasal administration of the peptides in a daily dose of 50 μg/kg resulted in an increase in the anticoagulant potential of the blood, namely, in an increase in the anticoagulant, fibrinolytic, and antiplatelet activity 20 h and 7 days after the last peptide injection. The arginine–containing peptide Arg–Pro–Gly–Pro had the most pronounced and stable effect on haemostasis under these experimental conditions.     

Epitope mapping of a monoclonal antibody directed against the α‐subunit of phosphofructokinase‐1 from Saccharomyces cerevisiae by screening phage display libraries

Phosphofructokinase‐1 from Saccharomyces cerevisiae is composed of two types of subunits, α and β. Subunit‐specific monoclonal antibodies were raised to elucidate structural and functional properties of both subunits. One monoclonal antibody, α‐F3, binds to an epitope either at the C‐terminal or at the N‐terminal part of the α‐polypeptide chain. By screening a heptapeptide library with this monoclonal antibody, a set of heptapeptides was selected, which contained the consensus sequences D–A–F and D–S–F. Two heptapeptides with these motifs were synthesized in order assess their capacity to inhibit the binding of antibody α‐F3 to native phosphofructokinase‐1. The peptide G–I–K–D–A–F–L inhibited the binding more strongly (IC₅₀ = 1.5 µM) than the peptide A–P–W–H–D–S–F (IC₅₀ = 33.3 µM). Sequence matching revealed the presence of the D–A–F motif in the polypeptide chain of phosphofructokinase‐1 at amino acid position 172–174. As a control, the nonapeptide A–P–T–S–K–D–A–F–L which corresponds to the sequence of the putative epitope was tested in the inhibition assay. In view of the high inhibitory capacity (IC₅₀ = 0.3 µM) it was concluded that this nonapeptide represents the continuous epitope of phosphofructokinase‐1 that is recognized by antibody α‐F3. Copyright © 1999 John Wiley & Sons, Ltd.     

N‐terminal diproline and charge group effects on the stabilization of helical conformation in alanine‐based short peptides: CD studies with water and methanol as solvent

Protein folding problem remains a formidable challenge as main chain, side chain and solvent interactions remain entangled and have been difficult to resolve. Alanine‐based short peptides are promising models to dissect protein folding initiation and propagation structurally as well as energetically. The effect of N‐terminal diproline and charged side chains is assessed on the stabilization of helical conformation in alanine‐based short peptides using circular dichroism (CD) with water and methanol as solvent. A1 (Ac–Pro–Pro–Ala–Lys–Ala–Lys–Ala–Lys–Ala–NH₂) is designed to assess the effect of N‐terminal homochiral diproline and lysine side chains to induce helical conformation. A2 (Ac–Pro–Pro–Glu–Glu–Ala–Ala–Lys–Lys–Ala–NH₂) and A3 (Ac–d Pro–Pro–Glu–Glu–Ala–Ala–Lys–Lys–Ala–NH₂) with N‐terminal homochiral and heterochiral diproline, respectively, are designed to assess the effect of Glu...Lys (i , i  + 4) salt bridge interactions on the stabilization of helical conformation. The CD spectra of A1 , A2 and A3 in water manifest different amplitudes of the observed polyproline II (PPII) signals, which indicate different conformational distributions of the polypeptide structure. The strong effect of solvent substitution from water to methanol is observed for the peptides, and CD spectra in methanol evidence A2 and A3 as helical folds. Temperature‐dependent CD spectra of A1 and A2 in water depict an isodichroic point reflecting coexistence of two conformations, PPII and β‐strand conformation, which is consistent with the previous studies. The results illuminate the effect of N‐terminal diproline and charged side chains in dictating the preferences for extended‐β, semi‐extended PPII and helical conformation in alanine‐based short peptides. The results of the present study will enhance our understanding on stabilization of helical conformation in short peptides and hence aid in the design of novel peptides with helical structures. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

Specific cleavage of beta-LPH and ACTH by tonin: release of an opiate-like peptide beta-LPH (61-78).

Tonin, a rat enzyme capable of cleaving angiotensinogen, the tetradecapeptide renin substrate and angiotensin I directly to antiotensin II is also shown to cleave beta-lipotropin into beta-LPH 1–50, 1–51, 51–60, 52–60, 61–78 and 79–91, thereby selectively releasing the opiate-like segment beta-LPH 61–78. Its action on ACTH was similar, releasing ACTH 1–8, 1–7, 3–8, 3–7 and 9–39. In both situations the cleavages are of a selective tryptic-chymotryptic type at specific arginine, phenylalanine residues. Comparison of the tonin cleavage with those of trypsin, trypsin in combination with citraconylation of the lysine residues of beta-LPH is made. The data presented show that tonin does not cleave Met-enkephalin and can be used as an enzyme to study the presence of endorphin-like sequences in polypeptides.     

Chromatography and Atomic Absorption Spectrometry for the Assessment of Heavy Metal Distribution among Amino Acids Used in Parenteral Nutrition Formulations—Studies with Cadmium and Lead

The distribution of Cd (II) and Pb (II) among amino acids in parenteral nutrition formulations was investigated by coupling ion-chromatography (HPLC/IC) and electrothermal atomic absorption spectrometry. The methodology was based on ion-exchange separation and fluorimetric amino acid detection after post-column derivatization. Cd (II) and Pb (II) were assayed in 500-µL fractions of the column effluent. The distribution of Cd (II) and Pb (II) in alanine (Ala), aspartic acid (Asp), glutamic acid (Glu), glycine (Gly), histidine (His), methionine (Met), phenylalanine (Phe), serine (Ser), and threonine (Thr) were analyzed by monitoring changes in the concentration of free amino acids by HPLC/IC. The results indicated that Cd (II) and Pb (II) were distributed according to the following trend: Gly–Cd?>?Gly–Pb?>?Ala–Cd?>?Ala–Pb?>?His–Cd?～?His–Pb?>?Thr–Cd?>?Thr–Pb?>?Phe–Cd?～?Phe–Pb?～?Asp–Cd?～?Asp–Pb?～?Met–Cd?～?Met–Pb?～?Glu–Cd?～?Glu–Pb?>?Ser–Cd?～?Ser–Pb. The effects of amino acid concentration and stability constants of amino acid–metal complexes are discussed.     

Biochemical Characterization of Different Molecular Forms of the Neural Cell Adhesion Molecule L1

The neural cell adhesion molecule L1 is a phosphorylated, integral membrane glycoprotein that is recovered from adult mouse brain tissue by immunoaffinity chromatography as a set of polypeptides with apparent molecular masses of 200, 180, 140, and 80 kilodaltons (L1–200, L1–180, L1–140, and L1–80, respectively). It has been shown that L1–140 and the phosphorylated L1–80 is generated from L1–200 by mild proteolytic treatment of intact cells. In the present study we have investigated the structural relationships between the different molecular forms of L1 and their location with regard to the surface membrane. We could show that L1–200 has two preferred cleavage sites, one that generates the amino terminal, extracellularly exposed L1–140 and the carboxy terminal L1–80 that spans the membrane. Cleavage at the other site leads to the generation of the amino terminally located L1–180 and the membrane-attached, phosphorylated carboxy terminal L1–30. This site is cleaved during treatment of live cultured cells with broad-spectrum, protease-free phospholipase C (but not phosphatidylinositol-specific phospholipase C) or exposure to sodium azide or cyanogen bromide. Other conditions that cause damage to cells do not lead to the generation of L1–180 and L1–30, suggesting a particular cell-intrinsic cleavage mechanism. L1–180 is truly soluble in aqueous solutions, since it can be recovered from culture supernatants and in the supernatant of a crude membrane fraction after incubation for 2 h at 37°C. Although trypsin treatment alone does not release L1–140 into the supernatant, combination of phospholipase C and mild tryptic treatment leads to the release of L1–140 and L1–50, the latter being most likely the extracellularly exposed domain of L1–80 that is complementary to the membrane-integrated phosphorylated L1–30. Phase separation experiments with Triton X-114 show that the released forms of L1–180 and L1–140 distribute into the aqueous phase, whereas they distribute into the detergent phase when in association with L1–200 or L1–80. However, when L1–80 is cleaved to yield the soluble L1–50 and membrane-anchored L1–30, L1–140 is released into the supernatant together with L1–50. A strong affinity of L1–200, L1–140, and L1–80 to each other is also indicated by the fact that they incorporate together into liposomes and separate only under strong detergent conditions. Also, a strong tendency to aggregate is observed for L1-containing liposomes, but not for those containing the adhesion molecules neural cell adhesion molecule and myelin-associated glycoprotein. Although the physiological roles of the soluble L1 forms, their mode of generation, and the strong affinity for each other remain to be investigated, the availability of soluble forms of L1 opens the possibility to use them as probes for the functional properties of L1 in assay systems involving live cells in vitro.     

Precipitation reconstruction for the southern Altay Mountains (China) from tree rings of Siberian spruce,reveals recent wetting trend

We developed six tree-ring width chronologies of Siberian spruce (Picea obovata) from the low elevation forest of the southern Altay Mountains in northern Xinjiang, China. Although the six chronologies come from different sampling sites, significant correlations existed among the chronologies (r ≥ 0.477), and the first principal component (PC1) accounted for 72.2% of total variance over their common period 1825–2010. Correlation response analysis revealed that radial growth of Siberian spruce is mainly limited by a 12-month precipitation starting from July of the previous year to June of the current year. We therefore developed a July–June precipitation reconstruction spanning 1825–2009, which explained 65.5% of the instrumental variance for the period 1962–2009. The information of our precipitation reconstruction suggested that dry conditions existed for the periods 1829–1838, 1852–1855, 1876–1888, 1898–1911, 1919–1923, 1932–1936, 1943–1955, 1963–1968, 1973–1984 and 2007–2009, and wet conditions for the periods AD 1825–1828, 1839–1851, 1856–1875, 1889–1897, 1912–1918, 1924–1931, 1937–1942, 1956–1962, 1969–1972 and 1985–2006. Spatial climate correlation analyses with gridded land surface data revealed that our precipitation reconstruction contains a strong precipitation signal for the Altay Mountain ranges. Our reconstruction agreed with the moisture-sensitive tree ring width series of Siberian larch from the Altay Mountains of Mongolia on a decadal timescale. In addition, in contrast to a drying trend in north central China, a clear wetting trend has occurred in the southern Altay Mountains since 1980s.     

Life Cycle of Isospora canis Nemeséri, 1959 in the Dog*

The life cycle of I. canis Nemeséri, 1959 was studied in experimentally infected dogs. Freshly sporulated oocysts were ovoid and 34–40 × 28–32 μm. The endogenous stages were found directly beneath the epithelium of the distal portion of the small intestinal villi. Most of the endogenous stages were in the lower 1/3 of the small intestine, but occasionally they were found in other portions of the small intestine. Three asexual generations were present. First-generation schizonts were 16–38 × 11–23 μm and contained 4–24 merozoites; mature 1st-generation merozoites were 8–11 × 3–5 μm. First-generation schizogony lasted up to 7 days after inoculation. Second-generation schizonts were 12–18 × 8–13 μm and contained up to 12 merozoites which were 11–13 × 3–5 μm. Second-generation schizogony was present on postinoculation days 6 and 7. Third-generation schizonts were formed by nuclear division of 2nd-generation merozoites. Most 2nd-generation merozoites underwent nuclear division without leaving the parasitophorous vacuole of the 2nd-generation schizont. Mature 3rd-generation schizonts were 13–38 × 8–24 μm and contained 6–72 merozoites. Third-generation merozoites were 8–13 × 1–3 μm. Third-generation schizogony was present on days 6–8 after inoculation. Mature macrogametes were 22–29 × 14–23 μm. Mature microgametocytes were 20–38 × 14–26 μm. Gametes were present on postinoculation days 7–10. Oocysts were present in tissue sections on postinoculation days 8–10 and 12. The prepatent period was 9–11 days.