Ciliary Membrane Proteins of Tetrahymena thermophila

SYNOPSIS SDS polyacrylamide gels of the ciliary membrane proteins of Tetrahymena thermophila revealed 5 major peaks and 11 minor protein peaks ranging in molecular weight from below 20,000 to above 250,000. The peaks resembled those found for ciliary membrane proteins of Paramecium aurelia. .     

Identification of Tetrahymena ciliary surface proteins labeled with sulfosuccinimidyl 6-(biotinamido) hexanoate and Concanavalin A and fractionated with Triton X-114.

Tetrahymena thermophila cells were labeled with sulfosuccinimidyl 6-(biotinamido) hexanoate, a sensitive nonradioactive probe for cell surface proteins, and Western blots of axonemes and ciliary membrane vesicles were compared to cilia fractionated with Triton X-114 (TX-114) in order to study the orientation of ciliary membrane proteins. Greater than 40 ciliary surface polypeptides, from greater than 350 kDa to less than 20 kDa, were resolved. The major surface 50-60 kDa proteins are hydrophobic and partition into the TX-114 detergent phase. Two high molecular weight proteins, one of which is biotinylated, comigrate with the heavy chains of ciliary dynein, sediment at 14S in a sucrose gradient, and partition into the TX-114 aqueous phase. Fractions containing these high molecular weight proteins as well as fractions enriched in 88-kDa and 66-kDa polypeptides contain Mg(2+)-ATPase activities. Detergent-solubilized tubulins partition into the TX-114 aqueous phase, are not biotinylated, and must not be exposed to the ciliary surface. The detergent-insoluble axoneme and membrane fraction contains a 36-kDa polypeptide and a portion of the 50-kDa polypeptides that otherwise partition into the detergent phase. These polypeptides could not be solubilized by ATP or by NaCl extraction and appear to be associated with pieces of ciliary membrane tightly linked to the axoneme. The ciliary membrane polypeptides were also tested for Concanavalin A binding and at least sixteen Con A-binding polypeptides were resolved. Of the major Con A-binding polypeptides, three are hydrophobic and partition into the TX-114 detergent phase, three partition into the TX-114 aqueous phase, and four partition exclusively in the detergent-insoluble fraction, which contains axonemes and detergent-resistant membrane vesicles.     

Tandem application of cationic colloidal silica and Triton X-114 for plasma membrane protein isolation and purification: towards developing an MDCK protein database

Plasma membrane (PM) proteins are attractive therapeutic targets because of their accessibility to drugs. Although genes encoding PM proteins represent 20-30% of eukaryotic genomes, a detailed characterisation of their encoded proteins is underrepresented, due, to their low copy number and the inherent difficulties in their isolation and purification as a consequence of their high hydrophobicity. We describe here a strategy that combines two orthogonal methods to isolate and purify PM proteins from Madin Darby canine kidney (MDCK) cells. In this two-step method, we first used cationic colloidal silica (CCS) to isolate adherent (Ad) and non-adherent (nAd) PM fractions, and then subjected each fraction to Triton X-114 (TX-114) phase partitioning to further enrich for hydrophobic proteins. While CCS alone identified 255/757 (34%) membrane proteins, CCS/TX-114 in combination yielded 453/745 (61%). Strikingly, of those proteins unique to CCS/TX-114, 277/393 (70%) had membrane annotation. Further characterisation of the CCS/TX-114 data set using Uniprot and transmembrane hidden Markov model revealed that 306/745 (41%) contained one or more transmembrane domains (TMDs), including proteins with 25 and 17 TMDs. Of the remaining proteins in the data set, 69/439 (16%) are known to contain lipid modifications. Of all membrane proteins identified, 93 had PM origin, including proteins that mediate cell adhesion, modulate transmembrane ion transport, and cell-cell communication. These studies reveal that the application of CCS to first isolate Ad and nAd PM fractions, followed by their detergent-phase TX-114 partitioning, to be a powerful method to isolate low-abundance PM proteins, and a useful adjunct for in-depth cell surface proteome analyses.     

Isolation and Identification of Novel ADP-Ribosylated Proteins from Streptomyces coelicolor A3(2)

An important role of protein ADP-ribosylation in bacterial morphogenesis has been proposed (J. Bacteriol. 178, 3785-3790; 178, 4935-4941). To clarify the detail of ADP-ribosylation, we identified a new kind of target protein for ADP-ribosylation in Streptomyces coelicolor A3(2) grown to the late growth phase. All four proteins (MalE, BldKB, a periplasmic protein for binding branched-chain amino-acids, and a periplasmic solute binding protein) were functionally similar and participated in the regulation of transport of metabolites or nutrients through the membrane. ADP-ribosylation was likely to occur on a cysteine residue, because the modification group was removed by mercuric chloride treatment. The modification site may be the site of lipoprotein modification necessary for protein export. This report is the first suggesting that certain proteins involved in membrane transport can be ADP-ribosylated.     

Morphological Alterations in Euglena gracilis Induced by Treatment with CTAB (Cetyltrimethylammonium Bromide) and Triton X-100: Correlations with Effects on Photophobic Behavioral Responses*

SYNOPSIS. Treatment of Euglena gracilis with the cationic detergent CTAB at concentrations of 0.05 mM or higher selectively inhibited the ability of the cells to respond with flagellar reorientation to a sudden decrease of light intensity (step-down photophobic response). The ability to respond similarly to an increase in light intensity (step-up photophobic response) was unaffected even at detergent concentrations at which the step-down response was completely inhibited. Electron microscopy of cells treated with 1.0 mM CTAB revealed selective destruction of the membrane of the reservoir and flagellum. No selective effects upon the step-down or step-up photophobic responses were found upon treatment of the cells with Triton X-100.     

Characterization of the T, L, I, S, M and P Cell Surface (Immobilization) Antigens of Tetrahymena thermophila: Molecular Weights, Isoforms, and Cross-Reactivity of Antisera

ABSTRACT. In the ciliate protist Tetrahymena thermophila the L, H, T, I, S, M and P cell surface proteins (immobilization antigens) are expressed under different conditions of temperature (L, H, T), culture media (I, S), and mutant genotype (M, P). Immunoblot and autoradiographic studies using antisera to purified protein show that the molecular weights of these proteins range from 25,000 to 59,000. The H, T, S, M and P antigens are recognized as single polypeptides, whereas L, I, and one allelic form of T each appear to consist of a family of polypeptides. Although antisera are specific in immobilization and immunofluorescence assays of surface protein in living cells, cross-reactivity is seen with denatured protein on immunoblots. It is hypothesized that the surface protein genes are organized into families of evolutionarily related isoloci.     

家蝇抗菌肽对细菌细胞表面特性影响及其作用机理的研究

利用微生物对十六烷吸附的方法(MATS方法)、微电泳方法与测定细菌质膜上β-半乳糖苷酶活性的方法,探讨了家蝇抗菌肽对大肠杆菌等6种细菌细胞表面特性及其细胞膜的作用机制。研究结果表明,抗菌肽使细菌表面电负性增强,对G 细菌细胞表面电荷的改变大于对G-的改变,使细菌细胞表面疏水性不同程度的下降。抗菌肽引起细菌细胞膜通透性迅速增加,不同细菌β-半乳糖苷酶释放的最大速度VP在3.86pmol/min～6.92pmol/min,相应的时间TP为0,由此推测抗菌肽对细胞膜的作用机制是“形成孔洞”。     

Purification and Characterization of a Major Cell Surface Glycoprotein in Zajdela Ascites Hepatoma Cells Which Displays a Potent Concanavalin A Receptor Activity

A major cell surface sialoglycoprotein with Concanavalin A receptor activity has been isolated from rat Zajdela ascites hepatoma cells. The sialic acid residues of the plasma membrane glycoproteins were specifically labeled by oxidation with NaIO₄ followed by reduction with NaB³H₄. Surface-labeled glycoproteins were released by short incubations with TPCK-trypsin at 37°C and then separated by gel filtration on Sepharose 6B column. The predominantly labeled fraction, GP II₂, was then purified by chromatography on DEAE-cellulose equilibrated with 0.05 M phosphate buffer, pH 7.5, and eluted with increasing molarities of NaCl. It was shown to be homogeneous by protein and carbohydrate staining on SDS-polyacrylamide gels, isoelectric focusing, rechromatography on DEAE-cellulose and immunoelectrophoresis. It has an apparent molecular weight of 110,000 daltons. The location of GP II₂on the cell surface was confirmed by the fact that it could be labeled metabolically with, D-(³H) glucosamine and externally through the nonpenetrating periodate-NaB³H₄ system. GP II₂could not be removed from the cell surface by high salt concentrations, chelator, or chaotropic agents but was released from the membrane by detergents. This suggests that GP II₂could be an integral protein. Analysis of the carbohydrate composition of GP II₂ revealed galactose, N-acetylglucosamine, N-acetylgalactosamine, and sialic acid as major constituents and mannose as a minor one. This suggests that it contains carbohydrate chains both O- and N-linked to the polypeptide chain, most of them being O-linked. Finally, GP II₂has a potent Concanavalin A receptor activity. It inhibits the interaction between Concanavalin A and hepatoma cells and suppresses its effects on hepatoma cell proliferation.     

Ciliary Rootlet Coiled-Coil 2 (crocc2) Is Associated with Evolutionary Divergence and Plasticity of Cichlid Jaw Shape

Cichlid fishes exhibit rapid, extensive, and replicative adaptive radiation in feeding morphology. Plasticity of the cichlid jaw has also been well documented, and this combination of iterative evolution and developmental plasticity has led to the proposition that the cichlid feeding apparatus represents a morphological “flexible stem.” Under this scenario, the fixation of environmentally sensitive genetic variation drives evolutionary divergence along a phenotypic axis established by the initial plastic response. Thus, if plasticity is predictable then so too should be the evolutionary response. We set out to explore these ideas at the molecular level by identifying genes that underlie both the evolution and plasticity of the cichlid jaw. As a first step, we fine-mapped an environment-specific quantitative trait loci for lower jaw shape in cichlids, and identified a nonsynonymous mutation in the ciliary rootlet coiled-coil 2 (crocc2), which encodes a major structural component of the primary cilium. Given that primary cilia play key roles in skeletal mechanosensing, we reasoned that this gene may confer its effects by regulating the sensitivity of bone to respond to mechanical input. Using both cichlids and zebrafish, we confirmed this prediction through a series of experiments targeting multiple levels of biological organization. Taken together, our results implicate crocc2 as a novel mediator of bone formation, plasticity, and evolution.     

Identification of Sporozoite Surface Proteins and Antigens of Eimeria nieschulzi (Apicomplexa)

Sodium dodecyl sulfate polyacrylamide gel electrophoresis, immunoblotting, lectin binding, and ¹²⁵I surface labeling of sporozoites were used to probe sporozoites of the rat coccidian, Eimeria nieschulzi. Analysis of silver stained gels revealed >50 bands. Surface iodination revealed about 14 well labeled, and about 10 weakly labeled but potential, surface proteins. The most heavily labeled surface proteins had molecular masses of 60, 53–54, 45, 28, 23–24, 17, 15, 14, 13, and 12 kD. Following electrophoresis and Western blotting, 2 of the 12 ¹²⁵I labeled lectin probes bound to two bands on the blots, which collectively indicated that two bands were glycosylated. Concanavalin A (ConA) specifically recognized a band at 53 kD, which may represent a surface glycoprotein, and a lectin derived from Osage orange (MPA) bound to a single band at 82–88 kD, that may also be a surface molecule. Immunoblotting using sera collected from rats inoculated orally with oocysts, as well as sera from mice hyperimmunized with sporozoites, revealed that many surface molecules appear to be immunogenic.     

Regulation of Plasma Membrane H+-Pump Activity by 14-3-3 Proteins in Barley (Hordeum disticum) Roots under Salt Stress

Regulatory changes in the activity of the plasma membrane H⁺-ATPase in salt-stressed roots were investigated using seven-day-old seedlings of two cultivars of barley (Hordeum disticum L.) with different salt tolerances: Moskovskii-121 (salt-tolerant) and Elf (salt-sensitive). During the first hour of salt stress, the rate of proton extrusion from the excised roots increased in parallel with the ATP hydrolase activity and the amount of 14-3-3 proteins bound to H⁺-ATPase in isolated plasma membranes. Subsequently, all these parameters decreased and dropped after 3–6 h below the initial levels. The initial stimulation of proton extrusion from the detached barley roots was caused by osmotic stress, whereas the subsequent retardation of proton extrusion was probably caused by a toxic effect of excessive Na⁺ content in the cytoplasm. The salt-stress responses showed similar trends in both cultivars, with the exception that Moskovskii-121 responded faster than cv. Elf. The results indicate that 14-3-3 proteins regulate the H⁺-ATPase activity in the plasma membranes of barley root cells during salt stress; furthermore, the response time might be a useful indicator to discriminate cultivars with different salt tolerances.     

Cell membrane enolase of Aedes albopictus C6/36 cells is involved in the entrance mechanism of dengue virus (DENV)

Currently, there are no antiviral drugs that effectively reduce the risks and treat the symptoms associated with dengue virus (DENV). Consequently, efforts remain primarily focused on transmission reduction. One such effort concerns DENV receptors in mosquito vectors Aedes aegypti and Aedes albopictus. Despite a lack of direct evidence demonstrating the binding of DENV to cells in mosquito vectors, one putative DENV binding protein has been α-enolase. To develop a deeper understanding, this study tested whether DENV proteins bind to enolase localized in the cytoplasmic membrane of C6/36 cells using both anti-enolase-specific antibodies, and by colocalization analysis, using confocal microscopy.Additionally, to probe the interaction of enolase with the DENV E protein, we performed a docking analysis using PatchDock and FireDock software packages. Study results demonstrate that the DENV E protein interacts with enolase in the plasma membrane of C6/36 cells of Ae. albopictus. Specific anti-enolase antibodies were found to inhibit DENV infection of these cells. Moreover, enolase was found to be localized to the cytoplasmic membrane, cytoplasm, and nucleus. These combined findings suggest that enolase participates in the entrance mechanism of DENV into vector cells.     

3-(Trifluoromethyl)-3-(m-isothiocyanophenyl)diazirine: Synthesis and chemical characterization of a heterobifunctional carbene-generating crosslinking reagent

A new hydrophobic heterobifunctional photocrosslinking reagent 3-(trifluoromethyl)-3-(m-isothiocyanophenyl)diazirine (TRIMID), a carbene precursor, and its radioiodinated analogue [¹²⁵I]TRIMID, have been synthesized and chemically characterized. The reagents were applied for membrane protein modification in human erythrocyte membranes and purple membranes fromHalobacterium halobium. Covalent labeling of the anion transport protein (band 3) via the isothiocyanate function was confirmed. Radiolabeled TRIMID was detected in at least two thermolysin-generated transmembrane fragments of the anion transport protein, and half-maximal inhibition of the erythrocyte anion transport activity was attained with 2.2 mM reagent. In bacteriorhodopsin (BR), a common binding site for the monofunctional phenylisothiocyanate and the bifunctional crosslinking reagent was identified: preincubation of purple membranes with TRIMID suppressed phenylisothio-[¹⁴C]-cyanate binding to BR. [¹²⁵I]TRIMID was recovered in V-1, the N-terminal segment of BR, which includes the phenylisothiocyanate binding site Lys-41. Light-induced intramolecular crosslinking of band 3-derived thermolytic fragments was not observed, although the carbene was generatedin situ and photocrosslinking of the protease V8 fragments of BR was not detected. Chemical and physicochemical characteristics of the new reagent are discussed with regard to limitations imposed for photoinduced site-directed crosslink formation.     

Two pools of Triton X-100-insoluble GABA(A) receptors are present in the brain, one associated to lipid rafts and another one to the post-synaptic GABAergic complex

Rat forebrain synaptosomes were extracted with Triton X-100 at 4 degrees C and the insoluble material, which is enriched in post-synaptic densities (PSDs), was subjected to sedimentation on a continuous sucrose gradient. Two pools of Triton X-100-insoluble gamma-aminobutyric acid type-A receptors (GABA(A)Rs) were identified: (i) a higher-density pool (rho = 1.10-1.15 mg/mL) of GABA(A)Rs that contains the gamma2 subunit (plus alpha and beta subunits) and that is associated to gephyrin and the GABAergic post-synaptic complex and (ii) a lower-density pool (rho = 1.06-1.09 mg/mL) of GABA(A)Rs associated to detergent-resistant membranes (DRMs) that contain alpha and beta subunits but not the gamma2 subunit. Some of these GABA(A)Rs contain the delta subunit. Two pools of GABA(A)Rs insoluble in Triton X-100 at 4 degrees C were also identified in cultured hippocampal neurons: (i) a GABA(A)R pool that forms clusters that co-localize with gephyrin and remains Triton X-100-insoluble after cholesterol depletion and (ii) a GABA(A)R pool that is diffusely distributed at the neuronal surface that can be induced to form GABA(A)R clusters by capping with an anti-alpha1 GABA(A)R subunit antibody and that becomes solubilized in Triton X-100 at 4 degrees C after cholesterol depletion. Thus, there is a pool of GABA(A)Rs associated to lipid rafts that is non-synaptic and that has a subunit composition different from that of the synaptic GABA(A)Rs. Some of the lipid raft-associated GABA(A)Rs might be involved in tonic inhibition.     

Consensus Sequence on the Genes Encoding the Major Outer Surface Proteins (OspA and OspB) of Borrelia garinii Isolate

Japanese Lyme borrelias classified as ribotype IV is predominant among isolates derived from clinical specimens, reservoir rodents and Ixodes persulcatus ticks, and has been characterized as Borrelia garinii. These B. garinii isolates have antigenic and genetic features apparently different from North American, European and other Asian isolates, especially in major outer surface proteins A (OspA) and B (OspB). In this study, we cloned and sequenced the genes encoding OspA and OspB from B. garinii strain FujiP2 (ribotype IV strain) isolated from I. persulcatus in Shizuoka, Japan. A sequence analysis revealed significant differences to the previously published sequences of ospA and ospB of B. burgdorferi sensu lato. The open reading frames of ospA and ospB consist of 822 and 888 nucleotides corresponding to the proteins of 273 and 295 amino acids, with molecular weights of 29,643 and 31,786 daltons, respectively. The most interesting finding is that the two osp genes share a consensus 282 bp sequence in their carboxy-terminal portions and that the ospB gene is flanked by a 282 bp-long direct repeat sequence. The deduced amino-acid (aa) sequences of OspA and OspB of strain FujiP2 showed 60.1% homology, and have overall similarities of 70.5%, 70.3% and 75.6% to OspAB proteins of B. burgdorferi sensu stricto strain B31, Borrelia afzelii strain ACA1 and Borrelia garinii strain Ip90, respectively.     

Coordination features of difunctionalized beta-cyclodextrins with carnosine: ESI-MS and spectroscopic investigations on 6A,6D-di-(beta-alanyl-L-histidine)-6A,6D-dideoxy-beta-cyclodextrin and 6A,6C-di-(beta-alanyl-L-histidine)-6A,6C-dideoxy-beta-cyclodextrin and their copper(II) complexes

The synthesis and characterization of two beta-cyclodextrins (beta-CD) functionalized with two units of carnosine (beta-alanyl-L-histidine) through the amino group, 6A,6C-(beta-alanyl-L-histidine)-6A,6C-dideoxy-beta-cyclodextrin (ACCDAH) and 6A,6D-(beta-alanyl-L-histidine)-6A,6D-dideoxy-beta-cyclodextrin (ADCDAH), are reported. NMR and C.D. data of the ligands indicate a different interaction of dipeptide chains with upper rim and cavity of beta-CD. Analogously, spectroscopic and electrospray ionization mass spectrometry data show different copper(II) complex species formed by the two regioisomers. The ability of carnosine-cyclodextrin derivatives to bind copper ions in a head-to-tail fashion induces the formation of oligomeric species (up to hexamers) in the case of ACCDAH, where the two carnosine moieties are adjacent, while in the ADCDAH case the mutual interaction between the peptidic chains of two ADCDAH molecules allows the almost exclusive formation of a copper-assisted self-assembled dimeric species.     

Investigation of the Axonal Transport of Three Acidic, Soluble Proteins (14-3-2, 14-3-3, and S-100) in the Rabbit Visual System

Abstract: The question of whether three acidic, water-soluble proteins (14-3-2, 14-3-3, and S-100, the first and last known to be brain-specific) are axonally transported was investigated in the rabbit visual system. The water-soluble proteins were obtained from individual optic nerves, combined optic tracts and lateral geniculate bodies, superior colliculi, and, in some instances, retinas at various times (1–56 days) after monocular injections of [³H]leucine. These proteins were separated by a two-step polyacrylamide gel electrophore-sis procedure that isolated 14-3-2, 14-3-3, and S-100 almost uncontaminated by other radioactivity. The isolated 14-3-2 and S-100 were demonstrated to be approx. 90% pure by a new method based on retarding the migration of these proteins by immunoadsorption during the first step of electrophoresis. An analysis of the radioactive labeling of the total soluble proteins (TSP) and the isolated acidic proteins revealed that: (1) S-100 was not axonally transported; (2) both 14-3-2 and 14-3-3 were part of one of the slow components of axonal transport (2-4 mm/day); (3) the radioactivity of 14-3-2 and 14-3-3 represented about 2.7% and 3.2%, respectively, of the radioactivity incorporated into the axonally transported TSP; (4) the ultimate distributions of the radioactively labeled 14-3-2 and 14-3-3 were the same (about 70% of each destined for the superior colliculus) and differed from that of the TSP; and (5) the rates of catabolism of the axonally transported 14-3-2 and 14-3-3 were slightly greater than that of the TSP, with half-lives for 14-3-2 and 14-3-3 estimated to be 11 and 10 days, respectively.     

Synthesis and in vitro evaluation of 3-(4-nitrophenyl)coumarin derivatives in tumor cell lines

Coumarins are naturally-occurring compounds that have attracted considerable interest due to their numerous biological activities depending on their pattern of substitution on the coumarin molecule. In this present investigation, we synthesized 3-(4-nitrophenyl)coumarin derivatives (9a–e) and evaluated their in vitro cytotoxic effect on human lung (A549), breast (MDA-MB-231) and prostate (PC3) cancer cell lines for 48 h using crystal violet dye binding assay. Cytotoxic effects of the most active compound on normal human lung (MRC-9) and breast (MCF-10A) cell lines, cell cycle analysis using flow cytometry and mitochondrial membrane potential (MMP) using Tetramethyl Rhodamine Methyl Ester (TMRM; rhodamine-123) fluorescent dye were also examined. Among the compounds that were evaluated, 9c showed cytotoxic effect (active), caused significant cells arrest (p < 0.05) in G₀/G₁ and S phases of cell cycle and loss of MMP in A459, MDA-MB-231 and PC3 cell lines. Additionally, the cytotoxic effect of 9c was compared to reference drugs (Coumarin and Docetaxel) for comparative study. These results further demonstrate that acetoxy group at C-7 and C-8 positions of 9c are responsible for the observed cytotoxic effect in these cancer cell lines.