Properties of E. coli of serological group 06:K13(L) isolated in acute intestinal diseases]

A study of 88 E. coli strains belonging to serological group 06: K13 (L) isolated from 50 children and 5 adults suffering from acute intestinal disturbances permitted to refer them to the serological types 06: K13 (L):H1 (82 strains) and 06: K13 (L): H--(6 strains), and to 5 biochemical types. Repeated isolation of E. coli belonging to serlogical group 06: K13 (L) in patients with acute intestinal diseases, and their absence in healthy children and adults indicated these microbes to be a possible etiological factor of the disease and to refer to the group group of enteropathogenic ones. Wide introduction of agglutinating sera 06: K13(L) into the work of practical laboratories is of expedience in connection with this.     

Genetic divergence between three sea urchin species of the genus Strongylocentrotus from the Sea of Japan

Intraspecific allozymic variation and interspecific genetic divergence were studied in three sea urchin species of the genus Strongylocentrotus (S. intermedius, S. nudus, S. pallidus) from the Sea of Japan. S. pallidus and S. intermedius showed high mean values of expected heterozygosity, H(e)=0.223+/-0.072 (17loci) and H(e)=0.230+/-0.065 (19loci), respectively. This estimate was somewhat lower in S. nudus, H(e)=0.126+/-0.043 (17loci). Estimates of Nei's genetic distance between S. nudus/S. intermedius (D=1.578, 17loci) and S. nudus/S. pallidus (D=1.327, 15loci) were considerably higher than that between S. intermedius/S. pallidus (D=0.269, 17loci). Invoking the protein clock hypothesis and using Panamanian geminate sea urchins for protein clock calibration, the time of divergence between S. intermedius and S. pallidus was estimated as 2.7MY. The results obtained for S. intermedius and S. nudus by us differ considerably from results obtained for these species by Norimasa Matsuoka and coworkers. The revealed discrepancies are discussed and the conclusion made that Matsuoka and coworkers' data on echinoderm biochemical genetics and systematics should be used with caution.     

Intestinal enterobacteria of the hibernating Apis mellifera mellifera L. bees

Dynamics of enterobacteria of normal intestinal microflora was studied in Apis mellifera mellifera L. bees hibernating under snow in the Western Urals. The cell numbers (N) of the predominant species Klebsiella oxytoca increased from 10-10(6) CFU/bee in November 2004 to 10(4)-10(7) CFU/bee in March 2005; its frequency of occurrence (P) increased from 92 to 100%. Increase of Providencia rettgeri (11.2004: N up to 10(6), P 25%; 03.2005: N 10(2)-10(6), P 80%) was accompanied by the substitution of Morganella morganii (11.2004: N up to 10(6), P 25%) with Proteus vulgaris (03.2005: N up to 10(5), P 8%). By spring, Hafnia alvei and Citrobacter sp., which are pathogenic to bees, disappeared (11.2004: N up to 10(5), P 13 and 10%, respectively). Endophytic species Pantoea agglomerans, Leclecria sp., and other representatives of the "Enterobacter agglomerans" group were present in November and after the first emergence in spring (N up to 10(5); November: P 15%; April: P 23%). In April, the number of enterobacteria decreased to 10(5), and P. rettgeri became the predominant species (P 54%) instead of K. oxytoca (P 43%).     

独活活性单体对中型指环虫的杀灭作用及其成分鉴定

利用硅胶柱层析和生物活性跟踪对独活乙醇提取物进行杀中型指环虫活性成分的分离纯化, 并测定活性成分在不同浓度条件下的离体生物活性, 通过薄层色谱(TLC)、紫外光谱(UV)、高效液相色谱(HPLC)、气质联用(GC-MS)对活性成分进行鉴定。结果表明: 从独活分离得到的F 和K1-2-3 两种流分均对中型指环虫具有不同程度的杀灭作用, 其中F 流分48h 内的完全杀灭中型指环虫的浓度为1.6 mg/L, 而K1-2-3 的使用浓度分别为5 mg/L 时, 其48h 内对中型指环虫的杀灭率为74.9%。经理化性质测定和光谱分析, F 流分和K1-2-3流分分别鉴定为蛇床子素和东莨菪内酯。       

Evidence for linkage between the swine L blood group and the loci specifying the receptors mediating adhesion of K88 Escherichia coli pilus antigens

Brush borders or enterocytes obtained from the small intestine of 248 pedigreed pigs were tested by adhesion assay in vitro with enterotoxigenic Escherichia (E.) coli strains, each expressing one of the three K88 pilus variants K88ab, K88ac and K88ad. All pigs were classified as belonging to one of the four adhesion phenotypes: I--K88ab(-), ac(-), ad(-); II--K88ab(-), ac(-), ad(+); III--K88ab(+), ac(+), ad(-); and IV--K88ab(+), ac(+), ad(+). Serum or red cells were typed for 15 blood group systems: A-O, B, C, D, E, F, G, H, I, J, K, L, M, N and O; for 11 biochemical polymorphisms: PI1, PI2, PO1A, A1BG, GPI, PGD, TF, HPX, ADA, PGM and AMY; the polymorphism at the IGHG1 locus. Linkage analysis was performed between the alleles at the locus (loci) specifying K88 receptors able to bind one or more different serological types of K88 E. coli and alleles for markers at other loci. Linkage was demonstrated between the locus for the L blood group system and the locus (loci) for K88 E. coli receptors (Z = 3.24), adding one locus (loci) to the previously identified linkage group IV (LGIV) [L-SLB]. The maximum likelihood estimate of the recombination fraction (theta) was 0.23. No evidence was found for linkage between any of the other biochemical and immunogenetic markers and the receptor locus (loci) of K88 E. coli.     

The UL45 gene product is required for herpes simplex virus type 1 glycoprotein B-induced fusion.

Herpes simplex virus type 1 (HSV-1) syncytial (syn) mutants cause formation of giant polykaryocytes and have been utilized to identify genes promoting or suppressing cell fusion. We previously described an HSV-1 recombinant, F1 (J.L. Goodman, M. L. Cook, F. Sederati, K. Izumi, and J. G. Stevens, J. Virol. 63:1153-1161, 1989), which has unique virulence properties and a syn mutation in the carboxy terminus of glycoprotein B (gB). We attempted to replace this single-base-pair syn mutation through cotransfection with a 379-bp PCR-generated fragment of wild-type gB. The nonsyncytial viruses isolated were shown by DNA sequencing not to have acquired the expected wild-type gB sequence. Instead, they had lost their cell-cell fusion properties because of alterations mapping to the UL45 gene. The mutant UL45 gene is one nonsyncytial derivative of F1, A4B, was found to have a deletion of a C at UL45 nucleotide 230, resulting in a predicted frame shift and termination at 92 rather than 172 amino acids. Northern (RNA) analysis showed that the mutant UL45 gene was normally transcribed. However, Western immunoblotting showed no detectable UL45 gene product from A4B or from another similarly isolated nonsyncytial F1 derivative, A61B, while another such virus, 1ACSS, expressed reduced amounts of UL45. When A4B was cotransfected with the wild-type UL45 gene, restoration of UL45 expression correlated with restoration of syncytium formation. Conversely, cloned DNA fragments containing the mutant A4B UL45 gene transferred the loss of cell-cell fusion to other gB syn mutants, rendering them UL45 negative and nonsyncytial. We conclude that normal UL45 expression is required to allow cell fusion induced by gB syn mutants and that the nonessential UL45 protein may play an important role as a mediator of fusion events during HSV-1 infection.     

Slow conformational exchange in DNA minihairpin loops: A conformationalstudy of the circular dumbbell d〈pCGC-TT-GCG-TT〉

In recent years various examples of highly stable two-residue hairpin loops (miniloops) in DNA have been encountered. As the detailed structure and stability of miniloops appear to be determined not only by the nature and sequence of the two bases in the loop, but also by the closing base pair, it is desirable to carry out in-depth studies of especially designed small model DNA compounds. Therefore, a circular DNA dumbbell-like molecule is tailored to consist of a stem of three Watson–Crick base pairs, flanked on each side by a minihairpin loop. The resulting circular DNA decanter 5′-d〈pCGC- TT-GCG- TT〉 -3′ ( I ) is studied in solution by means of nmr spectroscope. At a temperature of 269 K the molecule occurs in a 50/50 mixture of two dumbbell structures (denoted L2L2 and L2L4). L2L2 contains three Watson–Crick C-G base pairs and two two-residue loops (H2-family type) in opposite parts of the molecule. On raising the temperature from 269 to 314 K. The L2L4 conformer becomes increasingly dominant (95% at 314 K). This conformer has a partially disrupted closing G-C base pair in the 5′-GTTC-3′ loop with only one remaining solvent-accessible hydrogen bond between NH_α of the cytosine C(1) and O6 of the guanine G(8), whereas the opposite 5′-CTTG-3′ loop remains stable. The disruption of the C(1)-G(8) base pair in the L2L4 form is correlated with the presence of a syn orientation for the C(1) base at the 5′-3′ loop-stem junction in the 5′-GTTC-3′ loop. The two conformers. L2L2 and L2L4, occur in slow equilibrium (2–20 s^?1). Moderate line broadening of specific ¹H, ¹³C, and ³¹P resonances of residues C(1), G(8), T(9), and T(10) at low temperatures, due to chemical exchange between L2L2 and L2L4, show that the interconversion from an anti to syn conformer in residue C(1) has a small local effect on the structure of the dumbbell. T₁ relaxation measurements, chemical-shift considerations, and complete hand-shape calculations of the exchange process of the G(8) imino proton reveal a possibility for the existence of multiconformational slates in the anti–syn equilibrium. © 1995 John Wiley & Sons, Inc.     

Hydrophobic modulation of heme properties in heme protein maquettes

We have investigated the properties of the two hemes bound to histidine in the H10 positions of the uniquely structured apo form of the heme binding four-helix bundle protein maquette [H10H24-L6I,L13F](2), here called [I(6)F(13)H(24)](2) for the amino acids at positions 6 (I), 13 (F) and 24 (H), respectively. The primary structure of each alpha-helix, alpha-SH, in [I(6)F(13)H(24)](2) is Ac-CGGGEI(6)WKL.H(10)EEF(13)LKK.FEELLKL.H(24)EERLKK.L-CONH(2). In our nomenclature, [I(6)F(13)H(24)] represents the disulfide-bridged di-alpha-helical homodimer of this sequence, i.e., (alpha-SS-alpha), and [I(6)F(13)H(24)](2) represents the dimeric four helix bundle composed of two di-alpha-helical subunits, i.e., (alpha-SS-alpha)(2). We replaced the histidines at positions H24 in [I(6)F(13)H(24)](2) with hydrophobic amino acids incompetent for heme ligation. These maquette variants, [I(6)F(13)I(24)](2), [I(6)F(13)A(24)](2), and [I(6)F(13)F(24)](2), are distinguished from the tetraheme binding parent peptide, [I(6)F(13)H(24)](2), by a reduction in the heme:four-helix bundle stoichiometry from 4:1 to 2:1. Iterative redesign has identified phenylalanine as the optimal amino acid replacement for H24 in the context of apo state conformational specificity. Furthermore, the novel second generation diheme [I(6)F(13)F(24)](2) maquette was related to the first generation diheme [H10A24](2) prototype, [L(6)L(13)A(24)](2) in the present nomenclature, via a sequential path in sequence space to evaluate the effects of conservative hydrophobic amino acid changes on heme properties. Each of the disulfide-linked dipeptides studied was highly helical (>77% as determined from circular dichroism spectroscopy), self-associates in solution to form a dimer (as determined by size exclusion chromatography), is thermodynamically stable (-DeltaG(H)2(O) >18 kcal/mol), and possesses conformational specificity that NMR data indicate can vary from multistructured to single structured. Each peptide binds one heme with a dissociation constant, K(d1) value, tighter than 65 nM forming a series of monoheme maquettes. Addition of a second equivalent of heme results in heme binding with a K(d2) in the range of 35-800 nM forming the diheme maquette state. Single conservative amino acid changes between peptide sequences are responsible for up to 10-fold changes in K(d) values. The equilibrium reduction midpoint potential (E(m7.5)) determined in the monoheme state ranges from -156 to -210 mV vs SHE and in the diheme state ranges from -144 to -288 mV. An observed heme-heme electrostatic interaction (>70 mV) in the diheme state indicates a syn global topology of the di-alpha-helical monomers. The heme affinity and electrochemistry of the three H24 variants studied identify the tight binding sites (K(d1) and K(d2) values <200 nM) having the lower reduction midpoint potentials (E(m7.5) values of -155 and -260 mV) with the H10 bound hemes in the parent tetraheme state of [H10H24-L6I,L13F](2), here called [I(6)F(13)H(24)](2). The results of this study illustrate that conservative hydrophobic amino acid changes near the heme binding site can modulate the E(m) by up to +/-50 mV and the K(d) by an order of magnitude. Furthermore, the effects of multiple single amino acid changes on E(m) and K(d) do not appear to be additive.     

Production of mannitol and lactic acid by fermentation with Lactobacillus intermedius NRRL B-3693

Lactobacillus intermedius B-3693 was selected as a good producer of mannitol from fructose after screening 72 bacterial strains. The bacterium produced mannitol, lactic acid, and acetic acid from fructose in pH-controlled batch fermentation. Typical yields of mannitol, lactic acid, and acetic acid from 250 g/L fructose were 0.70, 0.16, and 0.12 g, respectively per g of fructose. The fermentation time was greatly dependent on fructose concentration but the product yields were not dependent on fructose level. Fed-batch fermentation decreased the time of maximum mannitol production from fructose (300 g/L) from 136 to 92 h. One-third of fructose could be replaced with glucose, maltose, galactose, mannose, raffinose, or starch with glucoamylase (simultaneous saccharification and fermentation), and two-thirds of fructose could be replaced with sucrose. L. intermedius B-3693 did not co-utilize lactose, cellobiose, glycerol, or xylose with fructose. It produced lactic acid and ethanol but no acetic acid from glucose. The bacterium produced 21.3 +/- 0.6 g lactic acid, 10.5 +/- 0.3 g acetic acid, and 4.7 +/- 0.0 g ethanol per L of fermentation broth from dilute acid (15% solids, 0.5% H(2)SO(4), 121 degrees C, 1 h) pretreated enzyme (cellulase, beta-glucosidase) saccharified corn fiber hydrolyzate.     

Structure of an oligodeoxynucleotide containing a butadiene oxide-derived N1 beta-hydroxyalkyl deoxyinosine adduct in the human N-ras codon 61 sequence

The solution structure of the N1-(1-hydroxy-3-buten-2(S)-yl)-2'-deoxyinosine adduct arising from the alkylation of adenine N1 by butadiene epoxide (BDO), followed by deamination to deoxyinosine, was determined, in the oligodeoxynucleotide d(CGGACXAGAAG).d(CTTCTCGTCCG). This oligodeoxynucleotide contained the BDO adduct at the second position of codon 61 of the human N-ras protooncogene, and was named the ras61 S-N1-BDO-(61,2) adduct. (1)H NMR revealed a weak C(5) H1' to X(6) H8 NOE, followed by an intense X(6) H8 to X(6) H1' NOE. Simultaneously, the X(6) H8 to X(6) H3' NOE was weak. The resonance arising from the T(17) imino proton was not observed. (1)H NOEs between the butadiene moiety and the DNA positioned the adduct in the major groove. Structural refinement based upon a total of 364 NOE-derived distance restraints yielded a structure in which the modified deoxyinosine was in the high syn conformation about the glycosyl bond, and T(17), the complementary nucleotide, was stacked into the helix, but not hydrogen bonded with the adducted inosine. The refined structure provided a plausible hypothesis as to why this N1 deoxyinosine adduct strongly coded for the incorporation of dCTP during trans lesion DNA replication, both in Escherichia coli [Rodriguez, D. A., Kowalczyk, A., Ward, J. B. J., Harris, C. M., Harris, T. M., and Lloyd, R. S. (2001) Environ. Mol. Mutagen. 38, 292-296], and in mammalian cells [Kanuri, M., Nechev, L. N., Tamura, P. J., Harris, C. M., Harris, T. M., and Lloyd, R. S. (2002) Chem. Res. Toxicol. 15, 1572-1580]. Rotation of the N1 deoxyinosine adduct into the high syn conformation may facilitate incorporation of dCTP via Hoogsteen-type templating with deoxyinosine, thus generating A-to-G mutations.     

Spasmolytic activity of some plants used by the Otomi Indians of Quéretaro (México) for the treatment of gastrointestinal disorders.

The present study was performed to investigate the effect of chloroform-methanol (1:1) extracts derived from Baccharis heterophylla H. B. K (Asteraceae), Desmodium grahami Gray (Leguminosae), Dyssodia pinnata var. pinnata Rob. (Asteraceae), Gentiana spathacea H. B. K. (Gentianaceae), Loeselia mexicana Brand. (Polemoniaceae), Selaginella pallescens Spring. (Selaginellaceae) and Zornia diphylla Pers. (Fabaceae) on the spontaneous contractions of isolated rat ileum. The concentration-response curves and IC50 values were obtained and the respective relaxant potencies were compared with that of the CHCl3-MeOH (1:1) extract of Datura lanosa. The data showed that all the extracts produce a concentration-dependent inhibition of spontaneous ileum contractions. In addition, the antimicrobial activity of the extracts against pathogenic enterobacteria was tested. The pharmacological actions shown by the extracts of the selected species tend to support ethnomedical use of the plants for the treatment of gastrointestinal disorders.     

重组水蛭素的突变及突变体部分性质研究

以基因突变结合动力学分析的方法研究了水蛭素空间结构及其与凝血酶的相互作用．采用基因定点突变和随机突变的方法得到两个重组水蛭素突变体,并从抗酰胺水解活性,抗凝血酶活力和稳定性三个方面,比较研究了重组水蛭素ｒＨＶ２中４７位和１１位两个氨基酸残基对其稳定性和抑制能力的影响．将ｒＨＶ２中Ｇｌｎ１１和Ａｓｎ４７分别突变为Ｈｉｓ１１和Ｌｙｓ４７后,ｒＨＶ２－Ｈ１１生物活力降低３０％,ｒＨＶ２－Ｋ４７生物活力提高６１％．测定抑制常数Ｋｉ表明,ｒＨＶ２－Ｈ１１突变体Ｋｉ值升高１４倍,ｒＨＶ２－Ｋ４７突变体Ｋｉ值降低１４倍,两个突变体的热稳定性均有所增强,ｒＨＶ２－Ｈ１１在酸性和碱性条件的稳定性降低．分析实验结果,可以认为：①４７位的Ｌｙｓ可能是通过氢键和静电两种作用力同时影响着水蛭素的三维结构和其与凝血酶的结合．②１１位氨基酸可能是水蛭素分子中另一个重要位点．     

中间球海胆、光棘球海胆及杂交F1代（中间球海胆♀×光棘球海胆♂）群体遗传多样性AFLP分析

应用AFLP技术对中间球海胆、光棘球海胆及杂交F1代（中间球海胆♀×光棘球海胆♂）群体的遗传多样性进行了分析。结果表明,4对引物共扩增得到272个位点,其中269个多态位点,总的多态位点比例为98.89%。3个群体的香农多样性指数分别为：0.2331±0.1273、0.2005±0.1385和0.2625±0.1067。群体内遗传相似度分别为：0.6876±0.0523、0.6501±0.0548和0.6552±0.0553。分子方差分析（AMOVA）结果表明,变异来源有25.39%来自群体间,有74.61%来自群体内,群体内的遗传多样性比较丰富。尽管杂交海胆在表型上可以明显分成两种类型,但是通过AFLP统计的遗传距离进行的个体聚类却随机聚在一起,不能分成两个群体。     

Self-assembly of heme A and heme B in a designed four-helix bundle: implications for a cytochrome c oxidase maquette

Heme A, a prosthetic group of cytochrome c oxidase [EC 1.9.3.1], has been introduced into two de novo designed four helix bundle proteins, [H10A24](2) and [H10H24](2), known to bind 2-4 equiv of heme B, respectively [Robertson, D. E., Farid, R. S., Moser, C. C., Mulholland, S. E., Pidikiti, R., Lear, J. D., Wand, A., J., DeGrado, W. F., and Dutton, P. L. (1994) Nature 368, 425-432]. [H10A24](2), [Ac-CGGGELWKL x HEELLKK x FEELLKL x AEERLKK x L-CONH(2)](2)(2), binds two heme A molecules per four-helix unit via bis-histidine ligation at the 10,10' positions with measured K(d) values of <0.1 and 5 nM, values much lower than those measured for heme B (K(d) values of 50 and 800 nM). The heme A-protein complex, [heme A-H10A24](2), exhibits well-defined absorption spectra in both the ferric and ferrous states, and an electron paramagnetic resonance spectrum characteristic of a low spin heme in the ferric form. A single midpoint redox potential (E(m8)) was determined for [heme A-H10A24](2) at -45 mV (vs SHE), which is significantly higher than that of the protein bound heme B (-130 and -200 mV). The observation of a single midpoint redox potential for [heme A-H10A24](2) and a pair of midpoints for [heme B-H10A24](2) indicates that the di-alpha-helical monomers are oriented in an anti topology (disulfides on opposite sides of bundle) in the former (lacking heme-heme electrostatic interaction) and syn in the latter. A mixture of global topologies was indicated by the potentiometric titration of the related [heme A-H10H24](2) which possess two distinct reduction potentials of +41 (31%) and -65 mV (69%). Self-assembly of the mixed cofactor heme A-heme B-[H10A24](2) was accomplished by addition of a single equivalent of each heme A and heme B to [H10A24](2). The single midpoint redox potential of heme B, E(m8) = -200 mV, together with the split midpoint redox potential of heme A in heme A-heme B-[H10A24](2), E(m8) = +28 mV (33%) and -65 mV (67%), indicated the existence of both syn and anti topologies of the two di-alpha-helical monomers in this four helix bundle. Synthesis of the mixed cofactor [heme A-heme B-H10H24](2) was accomplished by addition of a 2 equiv of each heme A and heme B to [H10H24](2) and potentiometry indicated the pair of hemes B resided in the 10,10' sites and heme A occupied the 24,24' sites. The results indicate that heme peripheral structure controls the orientation of the di-alpha-helical monomers in the four-helix bundle which are interchangeable between syn and anti topologies. In the reduced form, [heme A-H10A24](2), reacts quantitatively to form [carbonmonoxy-heme A-H10A24](2) as evidenced by optical spectroscopy. The synthetic [heme A-H10A24](2) can be enzymatically reduced by NAD(P)H with natural reductases under anaerobic conditions, and reversibly oxidized by dioxygen to the ferric form.     

Effect of uroguanylin on potassium and bicarbonate transport in rat renal tubules

The effect of uroguanylin (UGN) on K+ and H+ secretion in the renal tubules of the rat kidney was studied using in vivo stationary microperfusion. For the study of K+ secretion, a tubule was punctured to inject a column of FDC-green-colored Ringer's solution with 0.5 mmol KCl/L+/-10(-6) mol UGN/L, and oil was used to block fluid flow. K+ activity and transepithelial potential differences (PD) were measured with double microelectrodes (K+ ion-selective resin vs. reference) in the distal tubules of the same nephron. During perfusion, K+ activity rose exponentially, from 0.5 mmol/L to stationary concentration, allowing for the calculation of K+ secretion (JK). JK increased from 0.63+/-0.06 nmol.cm-2.s-1 in the control group to 0.85+/-0.06 in the UGN group (p<0.01). PD was -51.0+/-5.3 mV in the control group and -50.3+/-4.98 mV in the UGN group. In the presence of 10(-7) mol iberiotoxin/L, the UGN effect was abolished: JK was 0.37+/-0.038 nmol.cm-2.s-1 in the absence of, and 0.38+/-0.025 in the presence of, UGN, indicating its action on maxi-K channels. In another series of experiments, renal tubule acidification was studied, using a similar method: proximal and distal tubules were perfused with solutions containing 25 mmol NaHCO3/L. Acidification half-time was increased both in proximal and distal segments and, as a consequence, bicarbonate reabsorption decreased in the presence of UGN (in proximal tubules, from 2.40+/-0.26 to 1.56+/-0.21 nmol.cm-2.s-1). When the Na+/H+ exchanger was inhibited by 10(-4) mol hexamethylene amiloride (HMA)/L, the control and UGN groups were not significantly different. In the late distal tubule, after HMA, UGN significantly reduced JHCO3-, indicating an effect of UGN on H+-ATPase. These data show that UGN stimulated JK+ by acting on maxi-K channels, and decreased JHCO3- by acting on NHE3 in proximal and H+-ATPase in distal tubules.     

Structure of a ganglioside with Cad blood group antigen activity

The Cad antigen is a rare erythrocyte blood group antigen expressed on both sialoglycoprotein and ganglioside structures. It is related both serologically and biochemically to the Sda blood group antigen expressed on over 90% of Caucasian erythrocytes. We reported previously that Cad erythrocytes contain a novel ganglioside that binds Helix pomatia lectin and inhibits human anti-Sda antibody. We have now purified the Cad ganglioside and determined its structure. The ganglioside contained Glc-Gal-GlcNAc-GalNAc-NeuAc in a molar ratio of 1.00:1.94:0.95:0.93:1.05. Its chromatographic mobility was between that of GM1 and GD3. After treatment with beta-hexosaminidase (human placenta Hex A), the product migrated with 2-3-sialosylparagloboside (IV3NeuAcnLc4OseCer), it no longer bound H. pomatia lectin, and it acquired the ability to bind an antibody to sialosylparagloboside. Treatment of this material with neuraminidase (Vibrio cholerae) yielded a product with the mobility of paragloboside (nLc4OseCer) that bound monoclonal antibody 1B2, which is specific for terminal N-acetyllactosaminyl structures. Treatment of the Cad ganglioside with Arthrobacter ureafaciens neuraminidase yielded a product reactive with monoclonal antibody 2D4, which is specific for terminal GalNAc beta (1-4)Gal structures. These data provide strong evidence that the Cad ganglioside structure is GalNAc beta (1-4)[NeuAc alpha (2-3)]Gal beta (1-3)Gal beta (1-4)GlcCer. 1H NMR analysis also supports the conclusion that the terminal GalNAc is linked beta (1-4) to Gal. High-performance thin-layer chromatographic ganglioside patterns from three blood group Cad individuals showed a direct correlation between the quantity of Cad ganglioside and the strength of Cad antigen expression on the erythrocytes, as measured by hemagglutination.(ABSTRACT TRUNCATED AT 250 WORDS)     

Involvement of KCNQ2 subunits in [3H]dopamine release triggered by depolarization and pre-synaptic muscarinic receptor activation from rat striatal synaptosomes

KCNQ2 and KCNQ3 subunits encode for the muscarinic-regulated current (I(KM)), a sub-threshold voltage-dependent K+ current regulating neuronal excitability. In this study, we have investigated the involvement of I(KM) in dopamine (DA) release from rat striatal synaptosomes evoked by elevated extracellular K+ concentrations ([K+]e) and by muscarinic receptor activation. [3H]dopamine ([3H]DA) release triggered by 9 mmol/L [K+]e was inhibited by the I(KM) activator retigabine (0.01-30 micromol/L; Emax = 54.80 +/- 3.85%; IC50 = 0.50 +/- 0.36 micromol/L). The I(KM) blockers tetraethylammonium (0.1-3 mmol/L) and XE-991 (0.1-30 micromol/L) enhanced K+-evoked [3H]DA release and prevented retigabine-induced inhibition of depolarization-evoked [3H]DA release. Retigabine-induced inhibition of K+-evoked [3H]DA release was also abolished by synaptosomal entrapment of blocking anti-KCNQ2 polyclonal antibodies, an effect prevented by antibody pre-absorption with the KCNQ2 immunizing peptide. Furthermore, the cholinergic agonist oxotremorine (OXO) (1-300 micromol/L) potentiated 9 mmol/L [K+]e-evoked [3H]DA release (Emax = 155 +/- 9.50%; EC50 = 25 +/- 1.80 micromol/L). OXO (100 micromol/L)-induced [3H]DA release enhancement was competitively inhibited by pirenzepine (1-10 nmol/L) and abolished by the M3-preferring antagonist 4-diphenylacetoxy N-methylpiperidine methiodide (1 micromol/L), but was unaffected by the M1-selective antagonist MT-7 (10-100 nmol/L) or by Pertussis toxin (1.5-3 microg/mL), which uncouples M2- and M4-mediated responses. Finally, OXO-induced potentiation of depolarization-induced [3H]DA release was not additive to that produced by XE-991 (10 micromol/L), was unaffected by retigabine (10 micromol/L), and was abolished by synaptosomal entrapment of anti-KCNQ2 antibodies. Collectively, these findings indicate that, in rat striatal nerve endings, I(KM) channels containing KCNQ2 subunits regulate depolarization-induced DA release and that I(KM) suppression is involved in the reinforcement of depolarization-induced DA release triggered by the activation of pre-synaptic muscarinic heteroreceptors.     

Cyclic analogs of bradykinin, containing a carboxyl group]

1 alpha-beta-carboxypropionyl-cyclo(9----1 epsilon)-[Lys1, Gly6]bradykinin (Suc-c[Lys1, Gly6]B), 1 alpha-beta-carboxypropionyl-cyclo(10----1 epsilon)kallidin (Suc-cK), cyclo(10 gamma----1 epsilon)-[Glu10]kallidin (c[Glu10]K) and cyclo(11 gamma----1 epsilon)kallidylglutamic acid (cKG) were synthesized. Suc-c[Lys1, Gly6]B and Suc-cK were prepared by acylating the appropriate cyclopeptides with succinic anhydride. c[Glu10]K and cKG were obtained by the classic peptide synthesis, the cyclization being carried out with 61 and 42% yields, respectively. The protecting groups were then eliminated by catalytic hydrogenation. c[Glu10]K and cKG exerted myotropic action on isolated rat uterus (alpha 0.73 and 0.89, pD2 6.61 and 8.61, respectively). cKG displayed direct myotropic activity with respect to electrically stimulated rat vas deferens and guinea-pig ileum, potentiating the contractions (by 100%) in response to electric stimuli. c[Glu10]K and cKG elicit histamine release in isolated rat mast cells (EC30 4.91.10(-5) and 1.47.10(-6) M, respectively). Both cyclopeptides alter arterial pressure following intravenous administration to anaesthetized rats, cats and dogs and affect heart rate. In all assays cKG is more active than c[Glu10]K. Suc-c[Lys1, Gly6]B and Suc-cK do not possess myotropic, histamine-releasing or hypotensive activity, though they were found to elicit a transient increase of bloodflow in cats and dogs.     

Phylogenetic reassessment and biogeography of the Ectateus generic group (Coleoptera: Tenebrionidae: Platynotina)

Owing to the reinterpretation of its morphological synapomorphies, the taxonomic composition of the Ectateus generic group had been ambiguous. The present study scrutinized all existing taxonomic concepts of the group based on a cladistic analysis of the adult morphology of all of the Afrotropical platynotoid Platynotina genera. The phylogenetic relationships were reconstructed using parsimony and Bayesian inference. The results show that all previous taxonomic concepts of the Ectateus generic group concerned paraphyletic entities. The cladistic analysis revealed the following synapomorphies for the taxon: (1) presence of basal indentations of the pronotal disc, (2) ratio of prothorax width to its maximal height > 6.0, and (3) ratio of maximal height of the prothorax to total height < 0.3. Moreover, phylogenetic studies revealed the existence of the Upembarus generic group, a sister‐taxon group to the Ectateus generic group, within the Afrotropical platynotoid Platynotina. Autapomorphic and synapomorphic character mapping show that several taxonomic and nomenclatural changes are needed to consider the particular generic‐level entities traditionally assigned to Afrotropical platynotoid Platynotina as monophyletic lineages. The following taxonomic and nomenclatural adjustments are made in this paper: P teroselinus gen. nov. is erected to accommodate a single species that was previously assigned to Zidalus: Pteroselinus insularis comb. nov. Additionally, the following synonymies are proposed: Anchophthalmops (= Platykochius syn. nov. ), Angolositus (= Aberlencus syn. nov. , = Platymedvedevia syn. nov. ), Glyptopteryx (= Microselinus syn. nov. , = Quadrideres syn. nov. , = Synquadrideres syn. nov. ). In addition, Kochogaster is lowered in rank and is treated as one of the subgenera of Anchophthalmus. Moreover, Pseudoselinus is treated as a subgenus of Upembarus. An identification key to all Afrotropical platynotoid Platynotina genera and subgenera is presented. Zoogeographical analyses revealed the following dispersal barriers for the Ectateus generic group: (1) the Sahara (northern barrier); (2) the dry ecosystems of Botswana, Namibia, and South Africa (southern barrier); and (3) the Congolian rainforests (internal distributional gap). The ancestor of the taxon probably originated in East African ecoregions that predominantly contained wattletrees (acacias) and Commiphora Jacq. Moreover, past climate changes seem to have had a great impact on the observed generic distribution. © 2015 The Linnean Society of London