Vaccination of lactating dairy cows for the prevention of aflatoxin B1 carry over in milk

The potential of anaflatoxin B(1) (AnAFB(1)) conjugated to keyhole limpet hemocyanin (KLH) as a vaccine (AnAFB(1)-KLH) in controlling the carry over of the aflatoxin B(1) (AFB(1)) metabolite aflatoxin M(1) (AFM(1)) in cow milk is reported. AFB(1) is the most carcinogenic compound in food and foodstuffs amongst aflatoxins (AFs). AnAFB(1) is AFB(1) chemically modified as AFB(1)-1(O-carboxymethyl) oxime. In comparison to AFB(1), AnAFB(1) has proven to be non-toxic in vitro to human hepatocarcinoma cells and non mutagenic to Salmonella typhimurium strains. AnAFB(1)-KLH was used for immunization of cows proving to induce a long lasting titer of anti-AFB(1) IgG antibodies (Abs) which were cross reactive with AFB(1), AFG(1), and AFG(2). The elicited anti-AFB(1) Abs were able to hinder the secretion of AFM(1) into the milk of cows continuously fed with AFB(1). Vaccination of lactating animals with conjugated AnAFB(1) may represent a solution to the public hazard constituted by milk and cheese contaminated with AFs.     

低氧促进肺腺癌A549细胞迁移的机制*

目的: 探讨低氧促进肺腺癌A549细胞迁移的机制。方法: 培养肺腺癌A549细胞,转染慢病毒获得稳定敲低ACC1的A549细胞株,转染si-RNA获得敲低SREBP-1的A549细胞。分别以低氧(5％ O₂)联合低氧诱导因子1α(HIF-1α)抑制剂PX-478(25 μmol)处理A549细胞,低氧联合亚油酸(LA)(20 μmol)处理敲低ACC1的A549细胞,低氧处理敲低胆固醇调节原件结合蛋白1(SREBP-1)的A549细胞。Transwell实验检测细胞迁移,蛋白质印迹法检测HIF-1α、ACC1及上皮-间质转化(EMT)相关波形蛋白(Vimentin)及E-钙黏蛋白(E-Cadherin)的表达与SREBP-1的表达,实时荧光定量聚合酶链反应(RT-qPCR)检测低氧联合HIF-1α抑制剂PX-478(25 μmol)处理A549细胞后ACC1及SREBP-1 mRNA水平变化。每项实验重复三次。结果: 与常氧组相比,低氧组A549细胞迁移数增加,ACC1与HIF-1α表达上调(P均＜0.01),SREBP-1表达上调(P＜0.05);与低氧对照组相比,PX-478(25 μmol)抑制A549细胞迁移,SREBP-1表达下调(P＜0.05);低氧处理A549细胞后ACC1 mRNA上升(P＜0.05),SREBP-1 mRNA水平上升(P＜0.01);低氧并使用PX-478(25 μmol)处理A549细胞24 h,ACC1 mRNA水平下降(P＜0.05),SREBP-1 mRNA 水平下降(P＜0.01);转染si-RNA获得敲低SREBP-1的A549细胞,Transwell 实验显示si-SREBP-1组细胞迁移数较常氧对照组减少(P＜0.01);低氧处理si-SREBP-1组与si-NC组,与对照组相比si-SREBP-1组细胞迁移数减少(P＜0.01)但与常氧组相比差异无统计学意义(P＞0.05);Western blot检测到si-SREBP-1组ACC1表达较对照组下降(P＜0.01);低氧处理si-SREBP-1组,ACC1表达较对照组下降(P＜0.01);敲低ACC1抑制A549细胞迁移(P＜0.05),敲低ACC1后A549细胞在常氧和5％ O₂条件下细胞迁移数目差异无统计学意义(P＞ 0.05);低氧处理敲低ACC1的A549细胞并给予LA(25 μmol)促进A549细胞迁移(P＜0.05)。结论: 低氧通过HIF-1α/SREBP-1/ACC1途径调节脂肪酸代谢进而促进肺腺癌A549细胞迁移。     

Covalent structure of collagen: amino acid sequence of cyanogen bromide peptides from the amino-terminal segment of type III collagen of human liver

Human liver type III collagen was prepared by limited pepsin digestion, differential salt precipitation, and carboxymethylcellulose chromatography. Cyanogen bromide digestion of purified type III collagen chains yielded nine distinct peptides. Three peptides, alpha1(III)-CB3, alpha1(III)-CB7, and alpha1(III)-CB6, were isolated by carboxymethylcellulose chromatography and Sephadex G-50 SF gel filtration. Automated Edman degradation together with selective hydroxylamine cleavage and chymotrypsin and trypsin digestion enabled determination of their complete amino acid sequence. Compared with type I collagen, the data show tentative homology of alpha1(III)-CB3 with alpha1(I)-CB1, alpha1(I)-CB2, and alpha1(I)-CB4; alpha1(III)-CB7 with alpha1(I)-CB5; and alpha1(III)-CB6 with the amino-terminal portion of alpha1(I)-CB8. Close interspecies homology was found between the sequences presented here with 90 residues of alpha1(III)-CB3 and 26 of alpha1(III)-CB8 of calf aorta. The present study establishes the amino acid sequence of 229 residues near the amino terminus or nearly one-quarter of the type III collagen chains. The disaccharide, Glc-Gal, was convalently bound to hydroxylysine at a position corresponding to the same location in the alpha1(I) chain.     

Synthesis of structural elements of the capsular polysaccharides of Streptococcus pneumoniae types 6A and 6B

O-alpha-d-Glucopyranosyl-(1----3)-alpha, beta-L-rhamnopyranose (15), O-alpha-D-galactopyranosyl-(1----3)-O-alpha-D-glucopyranosyl-(1----3)-al pha, beta-L-rhamnopyranose (17), O-alpha-D-galactopyranosyl-(1----3)-O-alpha-D-glucopyranosyl-(1----3)- O-alpha-L-rhamnopyranosyl-(1----3)-D-ribitol (23), and O-alpha-D-galactopyranosyl-(1----3)-O-alpha-D-glucopyranosyl-(1----3)- O-alpha-L-rhamnopyranosyl-(1----4)-D-ribitol (27), which are structural elements of the capsular polysaccharides of Streptococcus pneumoniae types 6A and 6B ([----2)-alpha-D-Galp-(1----3)-alpha-D-Glcp-(1----3)-alpha-L-Rhap- (1----X)- D-Rib-ol-(5-P----]n; 6A X = 3, 6B X = 4), have been synthesised. Ethyl 3-O-allyl-2,4,6-tri-O-benzyl-1-thio-beta-D-glucopyranoside (3) was coupled with benzyl 2,4-di-O-benzyl-alpha-L-rhamnopyranoside (4), and subsequent deallylation (----14) and debenzylation gave 15. Condensation of 14 with ethyl 2,3,4,6-tetra-O-benzyl-1-thio-beta-D-galactopyranoside (2) followed by debenzylation gave 17. Acetylation of 17 followed by removal of AcO-1, conversion into the imidate, coupling with 1,2,4,5-tetra-O-benzyl-D-ribitol (11), deacetylation, and debenzylation gave 23. Coupling of the imidate with 1-O-allyloxycarbonyl-2,3,5-tri-O-benzyl-D-ribitol (12) followed by deallyloxycarbonylation, deacetylation, and debenzylation yielded 27.     

Chemical characterization of the immunomodulating polysaccharide of Aloe vera L

The polysaccharide isolated by alcohol precipitation of Aloe vera mucilaginous gel was found to have a Man:Glc:Gal:GalA:Fuc:Ara:Xyl ratio of 120:9:6:3:2:2:1 with traces of Rha and GlcA. Linkage analysis of the endo-(1-->4)-beta-d-mannanase-treated sample yielded Manp-(1--> (approximately 26%), 4-Manp (approximately 53%), 2,4-Manp (approximately 3%), 3,4-Manp (approximately 1%), 4,6-Manp (approximately 1%), 4-Glcp (approximately 5%), 4-Xylp (approximately 1%), Xylp-(1--> (approximately 2%), Galp-(1--> (approximately 5%), and traces of 4,6-Galp and 3,6-Galp. Hydrolysis with strong acids produced a mixture of short oligosaccharides and an acid-resistant fraction containing greater relative fractions of Manp-(1-->, Araf-(1-->, Xylp-(1-->, and 4-Xylp than the bulk polysaccharide. NMR analysis of oligosaccharides generated by endo-(1-->4)-beta-D-mannanase and acid hydrolysis showed the presence of di-, tri-, and tetrasaccharides of 4-beta-Manp, beta-Glcp-(1-->4)-Man, beta-Glcp-(1-->4)-beta-Manp-(1-->4)-Man, and beta-Manp-(1-->4)-[alpha-Galp-(1-->6)]-Man, consistent with a backbone containing alternating -->4)-beta-Manp-(1--> and -->4)-beta-Glcp-(1--> residues in a approximately 15:1 ratio. Analysis of the sample treated sequentially with endo-(1-->4)-beta-d-mannanase and alpha-D-galactosidase showed that the majority of alpha-Galp-(1--> residues were linked to O-2, O-3, or O-6 of -->4)-beta-Manp-(1--> residues, with approximately 16 -->4)-beta-Manp-(1--> residues between side chains. Our data provide direct evidence of a previously proposed glucomannan backbone, but draw into question previously proposed side-chain structures.     

Enzymatic synthesis of a novel cyclic pentasaccharide consisting of alpha-D-glucopyranose with 6-alpha-glucosyltransferase and 3-alpha-isomaltosyltransferase

A novel cyclic pentasaccharide (CPS) and a branched cyclic pentasaccharide (6G-CPS) consisting of d-glucopyranose were synthesized with 6-alpha-glucosyltransferase (6GT) and 3-alpha-isomaltosyltransferase (IMT) from Bacillus globisporus N75. The structure of CPS was cyclo-[-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->]. The other, 6G-CPS, had the structure cyclo-[-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-[alpha-D-Glcp-(1-->6)]-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->]. The formation of CPS was presumed to occur after the following four successive reactions: a 6-glucosyltransfer reaction with 6GT, a 4-glucosyltransfer reaction with 6GT, a 3-isomaltosyltransfer reaction with IMT, and a cyclization reaction with IMT.     

Kinetic properties of binding of myosin subfragment-1 with F-actin in the absence of nucleotide

The rate constant for the binding of myosin subfragment-1 (S-1) with F-actin in the absence of nucleotide, k1, and that for dissociation of the F-actin-myosin subfragment-1 complex (acto-S-1), k-1, were measured independently. The rate of S-1 binding with F-actin was measured from the time course of the change in the light scattering intensity after mixing S-1 with various concentrations of F-actin and k1 was found to be 2.55 X 10(6) M-1 X S-1 at 20 degrees C. The dissociation rate of acto-S-1 was determined using F-actin labeled with pyrenyl iodoacetamide (Pyr-FA). Pyr-FA, with its fluorescence decreased by binding with S-1, was mixed with acto-S-1 complex and the rate of displacement of F-actin by Pyr-FA was measured from the decrease in the Pyr-FA fluorescence intensity. The k-1 value was calculated to be 8.5 X 10(-3) S-1 (or 0.51 min-1). The value of the dissociation constant of S-1 from acto-S-1 complex, Kd, was calculated from Kd = k-1/k1 to be 3.3 X 10(-9) M at 20 degrees C. Kd was also measured at various temperatures (0-30 degrees C), and the thermodynamic parameters, delta G degree, delta H degree, and delta S degree, were estimated from the temperature dependence of Kd to be -11.3 kcal/mol, +2.5 kcal/mol, and +47 cal/deg . mol, respectively. Thus, the binding of the myosin head with F-actin was shown to be endothermic and entropy-driven.     

Immunochemical characterization and taxonomic evaluation of the O polysaccharides of the lipopolysaccharides of Pseudomonas syringae serogroup O1 strains

The O polysaccharide (OPS) of the lipopolysaccharide (LPS) of Pseudomonas syringae pv. atrofaciens IMV 7836 and some other strains that are classified in serogroup O1 was shown to be a novel linear alpha-D-rhamnan with the tetrasaccharide O repeat -->3)-alpha-D-Rhap-(1-->3)-alpha-D-Rhap-(1-->2)-alpha-D-R hap-(1-->2)- alpha-D-Rhap-(1--> (chemotype 1A). The same alpha-D-rhamnan serves as the backbone in branched OPSs with lateral (alpha1-->3)-linked D-Rhap, (beta1-->4)-linked D-GlcpNAc, and (alpha1-->4)-linked D-Fucf residues (chemotypes 1B, 1C, and 1D, respectively). Strains of chemotype 1C demonstrated variations resulting in a decrease of the degree of substitution of the backbone 1A with the lateral D-GlcNAc residue (chemotype 1C-1A), which may be described as branched regular left arrow over right arrow branched irregular --> linear OPS structure alterations (1Cleft arrow over right arrow 1C-1A --> 1A). Based on serological data, chemotype 1D was suggested to undergo a 1D left arrow over right arrow 1D-1A alteration, whereas chemotype 1B showed no alteration. A number of OPS backbone-specific monoclonal antibodies (MAbs), Ps(1-2)a, Ps(1-2)a(1), Ps1a, Ps1a(1), and Ps1a(2), as well as MAbs Ps1b, Ps1c, Ps1c(1), Ps1d, Ps(1-2)d, and Ps(1-2)d(1) specific to epitopes related to the lateral sugar substituents of the OPSs, were produced against P. syringae serogroup O1 strains. By using MAbs, some specific epitopes were inferred, serogroup O1 strains were serotyped in more detail, and thus, the serological classification scheme of P. syringae was improved. Screening with MAbs of about 800 strains representing all 56 known P. syringae pathovars showed that the strains classified in serogroup O1 were found among 15 pathovars and the strains with the linear OPSs of chemotype 1A were found among 9 of the 15 pathovars. A possible role for the LPS of P. syringae and related pseudomonads as a phylogenetic marker is discussed.     

Effect of nephrectomy and of adrenergic receptor blockers on the cardiorenal actions of endothelin

The cardiorenal actions of endothelin-1 (ET-1) were evaluated in rats following nephrectomy, in rats during alpha-adrenergic blockade with phentolamine, and in rats during beta-adrenergic blockade with propranolol. Female rats were anesthetized with pentobarbital and, following surgery, were allowed 60 min to stabilize before 3 x 20 min-control clearances were collected. ET-1 was then infused at a rate of 100 ng kg-1 min-1 for 30 min, the infusion was stopped, and three additional clearances were collected. Four groups of rats were studied: in Group 1 (n = 10), ET-1 was infused; in Group 2 (n = 5), a bilateral nephrectomy was performed 120 min before infusing ET-1; in Group 3 (n = 5), ET-1 was infused into rats treated with phentolamine (0.015 mg kg-1 min-1); and in Group 4 (n = 5), ET-1 was infused into rats treated with propranolol (0.015 mg kg-1 min-1). At 30 min during infusion of ET-1 into Group 1 rats, mean arterial blood pressure had increased (P less than 0.01) by 27 +/- 2% (SE) and the glomerular filtration rate had decreased (P less than 0.01) by 71 +/- 6% of baseline values. Nephrectomy potentiated and prolonged the ET-1-induced systemic vasoconstriction. Phentolamine had no effect on the cardiorenal actions of ET-1 whereas propranolol enhanced ET-1-induced changes in mean arterial blood pressure; mean arterial blood pressure increased 38 +/- 2% at 30 min during ET-1 + propranolol infusion (P less than 0.01 versus value with ET-1 alone). These data indicate that the kidney affects ET-1-induced systemic vasoconstriction and that beta-adrenergic (but not alpha-adrenergic) receptors are activated during infusion of ET-1 with a resultant attenuation of ET-1-induced changes in systemic blood pressure.     

Study of interaction of XRCC1 with DNA and proteins of base excision repair by photoaffinity labeling technique

The X-ray repair cross-complementing group 1 (XRCC1) protein plays a central role in base excision repair (BER) interacting with and modulating activity of key BER proteins. To estimate the influence of XRCC1 on interactions of BER proteins poly(ADP-ribose) polymerase 1 (PARP1), apurinic/apyrimidinic endonuclease 1 (APE1), flap endonuclease 1 (FEN1), and DNA polymerase beta (Pol beta) with DNA intermediates, photoaffinity labeling using different photoreactive DNA was carried out in the presence or absence of XRCC1. XRCC1 competes with APE1, FEN1, and PARP1 for DNA binding, while Pol beta increases the efficiency of XRCC1 modification. To study the interactions of XRCC1 with DNA and proteins at the initial stages of BER, DNA duplexes containing a photoreactive group in the template strand opposite the damage were designed. DNA duplexes with 8-oxoguanine or dihydrothymine opposite the photoreactive group were recognized and cleaved by specific DNA glycosylases (OGG1 or NTH1, correspondingly), although the rate of oxidized base excision in the photoreactive structures was lower than in normal substrates. XRCC1 does not display any specificity in recognition of DNA duplexes with damaged bases compared to regular DNA. A photoreactive group opposite a synthetic apurinic/apyrimidinic (AP) site (3-hydroxy-2-hydroxymethyltetrahydrofuran) weakly influences the incision efficiency of AP site analog by APE1. In the absence of magnesium ions, i.e. when incision of AP sites cannot occur, APE1 and XRCC1 compete for DNA binding when present together. However, in the presence of magnesium ions the level of XRCC1 modification increased upon APE1 addition, since APE1 creates nicked DNA duplex, which interacts with XRCC1 more efficiently.     

Synthesis and activity study of phosphonamidate dipeptides as potential inhibitors of VanX

In an effort to develop inhibitors of VanX, the phosphonamidate analogs of D-Ala-D-Ala dipeptides, N-[(1-aminoethyl) hydroxyphosphinyl]-glycine (1a), -alanine (1b), -valine (1c), -leucine (1d) and -phenylalanine (1e) were synthesized, characterized and evaluated using recombinant VanX. The crystal structure of the intermediate 6d was obtained (Deposition number: CCDC 839134), and structural analysis revealed that it is orthorhombic with a space group P2(1)2(1)2(1), the bond length of P-N is 1.62? and angle of C-N-P is 123.6°. Phosphonamidate 1(a-e) showed to be inhibitors of VanX with IC(50) values of 0.39, 0.70, 1.12, 2.82, and 4.13mM, respectively, which revealed that the inhibition activities of the phosphonamidates were dependent on the size of R-substituent of them, with the best inhibitor 1a having the smallest substituent. Also, 1a showed antibacterial activity against Staphylococcus aureus (ATCC 25923) with a MIC value of 0.25 μg/ml.     

Transient kinetics of the interaction of actin with myosin subfragment-1 in the absence of nucleotide.

The kinetics of the association of actin with myosin subfragment-1 (S1) has been studied by using S1 labeled at the sulfhydryl group SH1 with 5-(iodoacetamido)fluorescein (S1-AF). Upon rapid mixing in a stopped-flow apparatus, the fluorescence intensity of the fluorescein moiety increased by 50%, followed by a slower increase that was well resolved. This slow phase of the fluorescence change could not be fitted to either a monoexponential or a biexponential function, but it could be fitted to a sum of three exponential terms yielding three observed first-order rate constants (lambda i). The dissociation of acto.-(S1-AF) was studied by displacement of S1-AF from the complex with native S1. The dissociation kinetics was characterized by a single rate constant (approximately 0.012 s-1 at 20 degrees C), and this constant was independent of S1 concentration. Together with previous equilibrium data that were obtained under identified conditions for formation of acto-subfragment-1 (Lin, S.-H., and H. C. Cheung. 1991. Biochemistry. 30:4317-4323), a six-state two-pathway model is proposed as a minimum kinetic scheme for formation of rigor acto.S1. In this model, unbound subfragment-1 exists in two conformational states (S1' and S1) which are in equilibrium with each other, one corresponding to the previously established low-temperature state and the other to the high-temperature state. Each subfragment-1 state can interact with actin to form a collision complex, followed by two isomerizations to form two acto-subfragment-1 states (A.S1' and A.S1). Both isomerizations were visible in stopped-flow experiments. Two special cases of the model were considered: 1) a rapid pre-equilibration of the initial collision complex with actin and S1, and 2) trace accumulation of the collision complex. The first case required that the three combinations of the three observed rate constants be independent of actin concentration. The data were incompatible with this approximation. The other special case required that the sum of the lambda i vary linearly with actin concentration and the other two combinations of lambda i vary with actin concentration in a quadratic fashion. The present data were in agreement with the second case. At 20 degrees C and in 60 mM KCl, 2 mM MgCl2, 30 mM 2-([-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino)ethanesulfonic acid, and pH 7.5, the biomolecular association rate constants for the interaction of actin with S1' and S1 were 8.58 x 10(5) and 1.11 x 10(6) M-1 s-1, respectively.     

Structure of seven oligosaccharides excreted in the urine of a patient with Sandhoff's disease (GM2 gangliosidosis-variant O).

The urine of a patient with Sandhoff's disease (GM2 gangliosidosis-variant O) contains 10--12 N-acetylglucosamine-rich oligosaccharides in high amounts. The structures of seven of these have been determined: beta-GlcNAc(1--2)-alpha-Man-(1--3)-beta-man-(1--4)-GlcNAc; beta-GlcNAc-(1--4)-alpha-Man-(1--3)-beta-Man-(1--4)-GlcNAc; beta-GlcNAc-(1--2)-alpha-Man-(1--6)-beta-Man-(1--4)-GlcNAc; beta-GlcNAc-(1--4)-alpha-Man-(1--6)-beta-Man-(1--4)-GlcNAc; beta-GlcNAc-(1--2)-alpha-Man-(1--3)-[beta-GlcNAc-(1--2)-alpha-Man-(1--6)]beta-Man-(1--4)-GlcNAc; beta-GlcNAc-(1--2)-alpha-Man-(1--3)[beta-GlcNAc-(1--2)-alpha-Man-(1--6)][beta-GlcNAc-(1--4)]beta-Man-(1--4)-GlcNAc; beta-GlcNAc-(1--2)-alpha-Man(1)-(1--3)[beta-GlcNAc-(1--2)-alpha-Man(2)-(1--6)]beta-Man-(1--4)-GlcNAc, with additional beta-GlcNAc, with additional beta-GlcNAc-(1--4) on mannose (1) or (2). An unusual oligosaccharide, with a tri-branched beta-mannose, has been characterized as the major component excreted in urine.     

Dependence of the regulation of telomere length on the type of subtelomeric repeat in the yeast Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, chromosomes terminate with approximately 400 bp of a simple repeat poly(TG(1-3)). Based on the arrangement of subtelomeric X and Y' repeats, two types of yeast telomeres exist, those with both X and Y' (Y' telomeres) and those with only X (X telomeres). Mutations that result in abnormally short or abnormally long poly(TG(1-3)) tracts have been previously identified. In this study, we investigated telomere length in strains with two classes of mutations, one that resulted in short poly(TG(1-3)) tracts (tel1) and one that resulted in elongated tracts (pif1, rap1-17, rif1, or rif2). In the tel1 pif1 strain, Y' telomeres had about the same length as those in tel1 strains and X telomeres had lengths intermediate between those in tel1 and pif1 strains. Strains with either the tel1 rap1-17 or tel1 rif2 genotypes had short tracts for all chromosome ends examined, demonstrating that the telomere elongation characteristic of rap1-17 and rif2 strains is Tel1p-dependent. In strains of the tel1 rif1 or tel1 rif1 rif2 genotypes, telomeres with Y' repeats had short terminal tracts, whereas most of the X telomeres had long terminal tracts. These results demonstrate that the regulation of telomere length is different for X and Y' telomeres.     

Modulation of rap activity by direct interaction of Galpha(o) with Rap1 GTPase-activating protein.

We used the yeast two-hybrid system to identify proteins that interact directly with Galpha(o). Mutant-activated Galpha(o) was used as the bait to screen a cDNA library from chick dorsal root ganglion neurons. We found that Galpha(o) interacted with several proteins including Gz-GTPase-activating protein (Gz-GAP), a new RGS protein (RGS-17), a novel protein of unknown function (IP6), and Rap1GAP. This study focuses on Rap1GAP, which selectively interacts with Galpha(o) and Galpha(i) but not with Galpha(s) or Galpha(q). Rap1GAP interacts more avidly with the unactivated Galpha(o) as compared with the mutant (Q205L)-activated Galpha(o). When expressed in HEK-293 cells, unactivated Galpha(o) co-immunoprecipitates with the Rap1GAP. Expression of chick Rap1GAP in PC-12 cells inhibited activation of Rap1 by forskolin. When unactivated Galpha(o) was expressed, the amount of activated Rap1 was greatly increased. This effect was not observed with the Q205L-Galpha(o). Expression of unactivated Galpha(o) stimulated MAP-kinase (MAPK1/2) activity in a Rap1GAP-dependent manner. These results identify a novel function of Galpha(o), which in its resting state can sequester Rap1GAP thereby regulating Rap1 activity and consequently gating signal flow from Rap1 to MAPK1/2. Thus, activation of G(o) could modulate the Rap1 effects on a variety of cellular functions.     

The betagamma subunit of heterotrimeric G proteins interacts with RACK1 and two other WD repeat proteins

A yeast two-hybrid approach was used to discern possible new effectors for the betagamma subunit of heterotrimeric G proteins. Three of the clones isolated are structurally similar to Gbeta, each exhibiting the WD40 repeat motif. Two of these proteins, the receptor for activated C kinase 1 (RACK1) and the dynein intermediate chain, co-immunoprecipitate with Gbetagamma using an anti-Gbeta antibody. The third protein, AAH20044, has no known function; however, sequence analysis indicates that it is a WD40 repeat protein. Further investigation with RACK1 shows that it not only interacts with Gbeta(1)gamma(1) but also unexpectedly with the transducin heterotrimer Galpha(t)beta(1)gamma(1). Galpha(t) alone does not interact, but it must contribute to the interaction because the apparent EC(50) value of RACK1 for Galpha(t)beta(1)gamma(1) is 3-fold greater than that for Gbeta(1)gamma(1) (0.1 versus 0.3 microm). RACK1 is a scaffold that interacts with several proteins, among which are activated betaIIPKC and dynamin-1 (1). betaIIPKC and dynamin-1 compete with Gbeta(1)gamma(1) and Galpha(t)beta(1)gamma(1) for interaction with RACK1. These findings have several implications: 1) that WD40 repeat proteins may interact with each other; 2) that Gbetagamma interacts differently with RACK1 than with its other known effectors; and/or 3) that the G protein-RACK1 complex may constitute a signaling scaffold important for intracellular responses.     

Transient kinetics of the binding of ATP to actomyosin subfragment 1: evidence that the dissociation of actomyosin subfragment 1 by ATP leads to a new conformation of subfragment 1

The initial steps by which ATP dissociates and binds to actomyosin subfragment 1 (acto-SF-1) were studied. Two techniques were used: stopped-flow (for acto-SF-1 dissociation kinetics) and rapid-flow quench with ATP chase quenching (for ATP binding kinetics). The experiments were carried out in 40% ethylene glycol-5 mM KCI, pH 8, at 15 degrees C. Under these conditions, the binding of SF-1 to actin remains very tight. As with SF-1, the ATP chase technique could be used, first, to titrate active sites and, second, to study the kinetics of ATP binding to acto-SF-1. The kinetic constants obtained were compared with those of SF-1 alone and with the acto-SF-1 dissociation kinetics under identical conditions. The kinetics of the acto-SF-1 dissociation did not vary with the actin to SF-1 ratio, but the ATP binding kinetics did, and a maximum value was reached at a mole ratio of 2.5. At high ATP (100 microM), kdiss = 300 s-1, which compares with 49 s-1 and 13 s-1 for the ATP binding kinetics for acto-SF-1 (actin to SF-1 = 1:1) and SF-1, respectively. As with SF-1, the ATP binding to acto-SF-1 follows a hyperbolic law with the ATP concentration. This suggests a rapid equilibrium (K) followed by an essentially irreversible step (k).(ABSTRACT TRUNCATED AT 250 WORDS)     

Application of nano-probe NMR for structure determination of low nanomole amounts of arabinoxylan oligosaccharides fractionated by analytical HPAEC-PAD

A methodology for NMR analysis of low nanomole amounts of oligosaccharides fractionated by analytical HPAEC is presented. Arabinoxylan derived oligosaccharides purified by HPAEC-PAD on an analytical column, by single injections, were analyzed with nano-probe NMR and MALDI-TOF MS to provide full structural assignment. The NMR data were obtained with a 500 MHz NMR spectrometer equipped with a 1H-observe nano-probe. Both one- and two-dimensional experiments on arabinoxylan samples in the low nanomole range were performed, including 1H-1H DQF-COSY, 1H-1H TOCSY and 1H-1H ROESY. These experiments allowed, in combination with MALDI-TOF MS and literature NMR data, a complete structural determination of several tetra-, penta-, hexa- and heptasaccharides. Two new structures: alpha-L-Araf-(1 --> 2)-beta-D-Xylp-(1 --> 4)-beta-D-Xylp-(1 --> 4)-beta-D-Xylp-(1 --> 4)-D-Xylp and alpha-L-Araf-(1 --> 2)[alpha-L-Araf-(1 --> 3)]-beta-D-Xylp-(1 --> 4)-beta-D-Xylp-(1 --> 4)-beta-D-Xylp-(1 --> 4)-D-Xylp) were characterized, as well as some previously published structures.     

Interaction of isoforms of subfragment-1 of myosin, containing fluorescently labelled alkaline light chains, with muscle fiber actin

Using polarization microfluorimetry, the interaction of myosin subfragment 1 (S1) isoforms containing alkali light chains A1 and A2 respectively (S1(A1) and S1(A2] with F-actin of single glycerinated rabbit skeletal muscle fibers was studied. The alkali light chains of S1 were substituted by reassociation for A1 or A2 chains modified by a fluorescent label (1.5-IAEDANS) at the single SH-group located in the C-terminus. It was found that in S1(A1) bound to muscle fiber F-actin the mobility of the fluorescent label is lower than in S1(A2). At the same time the S1(A1) and S1(A2) interaction with F-actin induces similar changes in polarized fluorescence of rhodamine linked to falloidine which, in turn, is specifically bound to F-actin. It is concluded that the both S1 isoforms bind to F-actin and produce similar effects on the conformational state of actin filaments in muscle fibers. Local differences between S1(A1) and S1(A2) seem to be due to the interaction of the N-terminus of A1 within S1(A1) with the C-terminal region of actin.     

Regression by ACE inhibition of arteriosclerotic changes induced by chronic blockade of NO synthesis in rats

We previously reported that chronic inhibition of nitric oxide (NO) synthesis with N(omega)-nitro-L-arginine methyl ester (L-NAME) induces vascular inflammation at week 1 and produces subsequent arteriosclerosis at week 4 and that cotreatment with an angiotensin-converting enzyme (ACE) inhibitor prevents such changes. In the present study, we tested the hypothesis that treatment with an ACE inhibitor after development of vascular inflammation could inhibit arteriosclerosis in rats. Wistar-Kyoto rats were randomized to four groups: the control group received no drugs, the 4wL-NAME group received L-NAME (100 mg x kg(-1) x day(-1)) for 4 wk, the 1wL + 3wNT group received L-NAME for 1 wk and no treatment for the subsequent 3 wk, and the 1wL + 3wACEI group received L-NAME for 1 wk and the ACE inhibitor imidapril (20 mg x kg(-1) x day(-1)) for the subsequent 3 wk. After 4 wk, we observed significant arteriosclerosis of the coronary artery (medial thickening and fibrosis) and increased cardiac ACE activity in the 1wL + 3wNT group as well as in the 4wL-NAME group, but not in the 1wL + 3wACEI group. In a separate study, we examined apoptosis formation and found that posttreatment with imidapril (20 mg x kg(-1) x day(-1)) or an ANG II AT1-receptor antagonist, CS-866 (5 mg x kg(-1) x day(-1)), induced apoptosis (TdT-mediated nick end-labeling) in monocytes and myofibroblasts appearing in the inflammatory lesions associated with a clear degradation in the heart (DNA electrophoresis). In conclusion, treatment with the ACE inhibitor after 1 wk of L-NAME administration inhibited arteriosclerosis by inducing apoptosis in the cells with inflammatory lesions in this study, suggesting that increased ANG II activity inhibited apoptosis of the cells with inflammatory lesions and thus contributed to the development of arteriosclerosis.