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PubMed2004

Photomorphogenesis in the hypogeous fungus Tuber borchii: isolation and characterization of Tbwc-1, the homologue of the blue-light photoreceptor of Neurospora crassa.

Truffles form a group of plant-symbiotic Ascomycetes whose hypogeous life cycle is poorly understood. Here we present initial evidence for the influence of light on Tuber borchii mycelial growth and the identification and cloning of a gene, Tbwc-1, homologous to a blue-light photoreceptor of Neurospora crassa. Blue-light irradiation of T. borchii colonies inhibits their apical growth. It also alters apical growth in N. crassa. In Neurospora, the response is controlled by a nuclear photoreceptor, NcWC-1 (White Collar-1), which consists of a sensor domain (LOV) and a transcriptional factor moiety. We isolated a gene (Tbwc-1) whose deduced amino acid sequence shows a high similarity and colinearity of domains with NcWC-1, except for the polyglutamine regions. As previously found in Neurospora, Tbwc-1 mRNA is under light control and its steady state level increases upon irradiation. In silico analysis of the TbWC-1 sensor domain (LOV) supports the hypothesis that TbWC-1 is a photoreceptor, while the absence of the two polyglutamine regions involved in transcriptional activation in Neurospora suggests that this function in Tuber could be lost.

Amino Acid Sequence↗
PubMed1999

Beta1-integrins mediate Ca2+-signalling and T cell spreading via divergent pathways.

Interaction of Jurkat T-lymphocytes with two extracellular matrix (ECM) proteins of the basement membrane, laminin or collagen type IV, combined with poly-L-lysine resulted in a strong adhesion, a highly increased intracellular Ca2+-concentration ([Ca2]i), as compared to cells on laminin or collagen type IV alone and in spreading of the cells. The strong adhesion was independent of an increase in [Ca2+]i, was not mediated by a beta1-integrin, and was due to charge interaction between the positively charged polyaminoacid and the negatively charged cell surface. The latter was confirmed by substitution of poly-L-lysine by other positively charged polyaminoacids. In contrast, Ca+-signalling and spreading of the cells adhering to laminin or collagen type IV combined with poly-L-lysine was completely blocked by anti-beta1 mAb. However, spreading of the cells was independent of an increase in [Ca2+]i suggesting divergent signal transduction pathways leading to Ca2+-signalling and spreading of the cells. We elucidated these signal transduction pathways by inhibition of key enzymes involved. The tyrosine kinase inhibitor genistein blocked Ca2+-signalling as well as spreading, whereas inhibitors of PKC (calphostin C, GF109203x), PLCgamma (U73122) and PLA2 (bromophenacyl-bromide (BPB), 3-[4-octadecyl)benzoyl]acrylic acid (OBAA)) selectively blocked spreading of the cells.

Calcium Signaling↗
PubMed2005

Reversible inside-out micellization of pH-responsive and water-soluble vesicles based on polypeptide diblock copolymers.

The synthesis of a new zwitterionic diblock copolymer poly(l-glutamic acid)-b-poly(l-lysine) (PGA-b-PLys) is described, and its self-assembly into schizophrenic vesicles that can reversibly be produced in moderate acidic or basic aqueous solutions is reported. These pH-sensitive nanoparticles are expected to be very promising candidates in macromolecular nanobiotechnologies.

Hydrogen-Ion Concentration↗
PubMed1996

Mechanism of the thesaurismosis and altered lysosomal dynamics induced by poly-D-glutamic acid in kidney proximal tubular cells.

In the companion paper, we report that a single injection of poly-D-glutamic acid causes an acute lysosomal storage condition and apparently impairs the lysosomal fission dynamics. The present paper addresses the mechanisms of these two alterations using a combination of in vivo and in vitro biochemical approaches. After a single intravenous injection, 14C-poly-D-glutamic acid was rapidly cleared from the plasma and appeared in the urine. Yet, a small but sizable fraction of the injected polymer was taken up by the kidney cortex through a saturable process (Kuptake, 150 mg/kg body wt; uptakemax 96 micrograms/g cortex). Analytical subcellular fractionation of cortex homogenates demonstrated that at initial stages, the 14C label was predominantly associated with subcellular particles of intermediate size and low equilibrium density, and was therefore slowly transferred to larger particles equilibrating at high density, then codistributing with the lysosomal hydrolases. At a concentration of 10 mg/ml (equivalent to its estimated concentration in lysosomes), poly-D-glutamic acid formed micronic aggregates ( > or = 10 microns) when brought to solution at pH < or = 6 in relation to its decreased ionization (pKa of lateral chains approximately equal to 4.25). Finally, 1 day after the injection of poly-D-glutamic acid, the activities of several lysosomal enzymes (hexosaminidase, cathepsin B, acid sphingomyelinase, and sulfatase B), but not of all of them (eg, acid phosphatase), were increased in the kidney cortex. We propose that poly-D-glutamic acid reaches lysosomes by adsorptive endocytosis and becomes concentrated within these organelles because its withstands hydrolysis until it forms aggregates or precipitates, causing a decrease in the fluidity or the deformability ("gelling") of the lysosomal matrix. This should alter the dynamics of intercommunication of these organelles by impairing their fission without a proportionate effect on their fusion properties. In addition, the data suggest that the presence of poly-D-glutamic acid directly or indirectly slows down the degradation of several lysosomal enzymes.

Animals↗
PubMed1980

Retardation of calcium oxalate formation by polyacidic peptides.

The polycarboxylic amino acids were examined as possible retarding agents in the precipitation of calcium oxalate. The effect was observed by continuous determination of the concentration of calcium ions in a mimic urine solution. Both polyglutamic and polyaspartic acids, when used in 5 to 100 ppm concentrations, significantly retarded calcium oxalate precipitation.

Aspartic Acid↗
PubMed2000

Observation of tetrahydrofolylpolyglutamic acid in bacteria cells by matrix-assisted laser desorption/ionization mass spectrometry.

Tetrahydrofolylpolyglutamic acid in whole bacteria cells and cell lysates is analyzed by matrix-assisted laser desorption/ionization mass spectrometry. The speed, mass information, and tolerance to impurities of this technique make it ideal for monitoring the glutamation levels of folic acid in biological systems. Folylpolyglutamic acid is observed in a few strains of E. coli and two species of Staphylococcus bacteria. The effects of growth time, growth media, and the addition of methotrexate, a dihydrofolate reductase inhibitor, are also studied.

Antimetabolites, Antineoplastic↗
PubMed1984

Antitumor agent poly (amino acid) conjugates as a drug carrier in cancer chemotherapy.

Antitumor agent melphalan was conjugated through a carbodiimide-catalyzed reaction to poly (L-lysine) (71.3K and 2K) and poly (L-glutamic acid) (60K and 14K) at a ratio of approximately one molecule per 7-lysyl and 23-glutamate residues, respectively. These conjugates had 40-70% of alkylating activities by themselves in vitro as compared with free melphalan. Poly (glutamic acid) conjugates showed the antitumor activity in vivo against Yoshida sarcoma in rats. In addition, poly (glutamic acid) conjugates containing 3H-phenylalanine which was used as a model compound instead of melphalan had a sustained release property of radioactivity and the release rates could be regulated by exopeptidases. After subcutaneous injection as the first choice of routes of administration, moreover, the conjugates were found to be absorbed through the lymphatic transport system, probably due to the macromolecularity. These results suggest that the melphalan-poly (amino acid) conjugates are one of good candidates to attain the sustained release and targeting of drugs in cancer chemotherapy.

Absorption↗
PubMed2004

Ionic strength-dependent pK shift in the helix-coil transition of grafted poly(L-glutamic acid) layers analyzed by electrokinetic and ellipsometric measurements.

Surface-bound layers of poly(L-glutamic acid) prepared by a recently described "grafting-from" method were analyzed with respect to electrical charging and structural alterations upon variation of pH and concentration of the background electrolyte in aqueous solutions. The microslit electrokinetic setup (MES) was utilized for the combined determination of zeta potential and surface conductivity on the basis of streaming potential and streaming current measurements at polypeptide layers in contact with aqueous electrolyte solutions of varied composition. In situ ellipsometry was applied at similar samples immersed in identical aqueous solutions to investigate the influence of the solution pH on the structure of the polypeptide layers. Zeta potential and Dukhin number versus pH plots revealed the dissociation behavior of the surface-bound polypeptides indicating a significant shift of the pK of their acidic side chains correlating with the concentration of the background electrolyte potassium chloride and the related variation of the Debye screening length. Surface conductivity data pointed at a more expanded structure of the polypeptide layer in the fully dissociated state as an increased ion conductance in this part of the interface was determined. The occurrence of a strong increase of the thickness and a corresponding decrease of the refractive index for the coil state of the layer strongly supports the findings of the electrokinetic measurements. This fully reversible "switching" of the layer structure was attributed to helix-coil transitions within the grafted polypeptides induced by the dissociation of carboxylic acid functions of the polypeptide side chains. The shift of the "switching pH" of the surface-bound poly(L-glutamic acid) layers at varied concentrations of the background electrolyte was interpreted as a result of the pK shift of the carboxylic acid groups of the polypeptide side chains. The observed patterns prove that the electrostatic interactions causing this shift occur within but not between the grafted chains.

Electrochemistry↗
PubMedDate not supplied

[Acid polypeptides as intensifiers of the formation of irreparable double-stranded DNA breaks induced by the gamma irradiation of mammalian cells].

Acid polypeptides, synthetic analogues of a natural modifier of lethal effect of radiation, were shown to inhibit double-strand DNA breaks (DSB) repair, to increase irreparable DSB accumulation and to enhance the formation of structural chromosome rearrangements in gamma-irradiated mammalian cells. The authors discuss the possibility of involvement of proteins, that contain amino acid sequences comparable, in length, with a modifier, into radiation formation of irreparable DSB.

Animals↗
PubMed2002

Molecular chaperones enhance the degradation of expanded polyglutamine repeat androgen receptor in a cellular model of spinal and bulbar muscular atrophy.

Spinal and bulbar muscular atrophy (SBMA) is one of a growing number of neurodegenerative diseases caused by a polyglutamine-encoding CAG trinucleotide repeat expansion, and is caused by an expansion within exon 1 of the androgen receptor (AR) gene. The family of polyglutamine diseases is characterized by the presence of ubiquitinated, intranuclear inclusions associated with molecular chaperones and 26S proteasome components, although the role of these inclusions in the pathogenesis of polyglutamine diseases remains unclear. The over-expression of molecular chaperones of the Hsp70 and Hsp40 families has been shown to modulate inclusion frequency and cellular toxicity. We developed a cell culture system which enables the quantitative analysis of the effects of molecular chaperones on the biochemical properties of an expanded repeat AR. Using this approach, we demonstrate that Hsp70 and its co-chaperone Hsp40 not only increase expanded repeat AR solubility, but function to enhance the degradation of expanded repeat AR through the proteasome. Furthermore, our studies indicate that these molecular chaperones significantly decrease the half-life of an expanded repeat AR. Molecular chaperone enhancement of protein degradation points to the modulation of molecular chaperones as a potential therapeutic target for polyglutamine diseases.

Acetylcysteine↗
PubMed2005

Circular dichroism spectroscopic study of non-covalent interactions of poly-L-glutamic acid with a porphyrin derivative in aqueous solutions.

The interactions of poly-L-glutamic acid and a cationic porphyrin derivative in aqueous solutions were studied by the combination of vibrational circular dichroism (VCD) and electronic circular dichroism (ECD) spectroscopies. It was found that non-covalent interactions between both agents influence the structure of the polymeric matrix and the guest porphyrins and vice versa, but the physico-chemical properties of the solutions, especially the pH and the relative permittivity of the solvent, play a key role in the structure of the polypeptide part of the formed complexes. It was shown that the interaction with porphyrins prevents the precipitation of poly-L-glutamic acid in aqueous solution at acidic pH. In special conditions, the porphyrins attached to the polypeptide probably possess face-to-face interaction as demonstrated by the enhancement of the characteristic ECD signal and the appearance of sidebands on its short and long wavelength sides.

Circular Dichroism↗
PubMedDate not supplied

Structural characterization of self-assembled polypeptide films on titanium and glass surfaces by atomic force microscopy.

Chemically modified biomaterial surfaces (titanium and glass) covered with polyelectrolyte self-assembled films formed by the alternating adsorption of cationic poly-L-lysine (PLL) and anionic poly-L-glutamic acid (PGA) were structurally characterized by atomic force microscopy. Complementary information concerning the thickness and layer-by-layer growth of the films was provided by optical waveguide light-mode spectroscopy. The frequently used ex situ and the rarely used in situ build-up methods were compared. Important aspects of the industrial applicability of these films, their stability in time, and possible differences in their morphology were investigated. The films revealed a granular pattern, with grain diameters of 270 +/- 87 nm for glass (up to 8 bilayers) and 303 +/- 89 nm for titanium (up to 10 bilayers), independently of the build-up procedure. Both surfaces displayed a rehydration capability, the titanium surface exhibiting a better stability in time. The high roughness values observed at acidic or basic pH are related to the degree of ionization of PGA and PLL.

Adsorption↗
PubMed2003

Side-chain dynamics of an alpha-helical polypeptide monitored by fluorescence.

The "blob" model, developed to analyze the fluorescence decays of polymers randomly labeled with pyrene, has been applied to a series of pyrene-labeled poly(glutamic acid)s (PyPGA) in DMF and carbonated buffer solutions at pH 9. Poly(glutamic acid) (PGA) exists in the ionized form in the buffer solutions as poly(sodium glutamate) (PGNa). PGA adopts an alpha-helical conformation in DMF, whereas in aqueous solution PGNa is a random coil. Fluorescence, UV-vis absorption, and circular dichroism measurements indicate that in our studies pyrene pendants attached themselves along PGA in a clustered manner. Simulations were carried out to establish that the geometry of the PGA alpha-helix induces the high level of pyrene clustering. Since the level of pyrene clustering decreased with lower pyrene content, information about naked PGA was retrieved by extrapolating the trends obtained by fluorescence to zero pyrene content. Analysis of the fluorescence decays demonstrated that during its lifetime an excited pyrene probes a 32 amino acid section of the PGA alpha-helix. This result was supported by molecular mechanics optimizations. This study establishes that the blob model, originally used to monitor the encounters between pyrenes attached randomly onto a polymer adopting a random coil conformation, can also be applied to study the dynamics of the side chains of structured proteins. Since the blob model helps in monitoring the encounters between amino acids in the initial state (i.e., random coil) and in the final state (i.e., structured protein) of the folding pathway of a protein, it could be applicable to the study of protein folding.

Circular Dichroism↗
PubMed1998

Apatite precipitation after incubation of biphasic calcium-phosphate ceramic in various solutions: influence of seed species and proteins.

The dissolution-precipitation process for calcium-phosphate ceramics in contact with biological fluid was studied by incubating blocks of biphasic calcium phosphate composed of hydroxyapatite (HA) and beta-tricalcium phosphate (beta-TCP) in different solutions: ionic simulated body fluid (SBF) without protein or SBF that contained various proteins and macromolecules separately (fibronectin, vitronectin, albumin, and poly-L-glutamic acid). Transmission electron microscopy studies revealed that apatite-precipitated microcrystals appeared around ceramic crystals as a result of secondary nucleation; microcrystals were in continuity with the lattice planes of the HA crystals but in a different direction from that of beta-TCP; the size of the precipitates was smaller when fibronectin, vitronectin, and poly-(L-glutamic acid) were present in SBF as compared to SBF without protein; and fibronectin and vitronectin initiated crystal nucleation in the void spaces between the ceramic crystals.

Apatites↗
PubMedDate not supplied

Functional expression of a microinjected Ed alpha gene in C57BL/6 transgenic mice.

The class II major histocompatibility antigens, I-A and I-E, have been detected on the surface of certain immunocompetent cells, including B lymphocytes and monocytes. These molecules are involved in cell-cell interactions in the immune responses. Each class II antigen consists of two subunits, alpha and beta chains, and the genes encoding these subunits have been well characterized at the molecular level. To analyse the regulatory mechanism of E alpha gene expression and the role of the I-E antigen in the regulation of the immune responses, we have produced transgenic mice by microinjecting cloned Ed alpha genes into fertilized eggs of C57BL/6 mice of b haplotype. This strain of mouse carries a deletion in the upstream (5') region of the E alpha gene covering the transcriptional promoter and, therefore, does not express this gene. Interestingly, this genetic defect of the E alpha gene is accompanied by the inability of the host mouse to respond to a certain set of antigens, phenomena generally termed Ir gene control. We report here that the Ed alpha genes are expressed in these transgenic mice to form the I-Ed alpha Eb beta antigen on the surface of B lymphocytes and monocytes and that these I-E antigens are functional in terms of the induction of a mixed lymphocyte reaction and the restoration of immune responsiveness to poly(L-glutamic acid-L-lysine-L-phenylalanine) (GL-Phe).

Animals↗
PubMed2000

Applicability of broken-rodlike chain model to conformational analysis of polypeptide chain.

In order to check the applicability of the broken-rodlike (BR) chain model, consisting of several rods alternatively joined by flexible random coils, to the conformational analysis of a polypeptide chain in the helix-to-coil transition regions, two relations predicted by the Zimm and Bragg theory and the method with the BR chain model are compared. It is shown that, despite a clear difference between the models employed in the two methods, they give substantially identical results in both probability P(j) that a helical residue is in a helical sequence j units long and averaged helical fraction dependence of the mean-squared radius of gyration. Thus the use of the method with the BR chain model in the conformational analysis of a polypeptide chain could be rationalized, at least, with the same degree of approximation as is assumed in the Zimm and Bragg theory. Using the scattering function for the BR chain model, averaged helical-sequence lengths are evaluated for partially ionized poly(L-glutamic acid) (PGA) in added-salt aqueous solution and nonionized PGA in N-methylacetamide, both in a helical state. As a result, it is shown that the length in the latter molecule is approximately tenfold longer than that in the former one.

Computer Simulation↗
PubMedDate not supplied

Versatile procedure of multiple introduction of 8-aminomethylene blue into oligonucleotides.

The coupling of 8-aminomethylene blue to oligonucleotides via poly-L-glutamic acid linker using carboxy-anchor groups will be described. The introduction of carboxy-anchor groups into oligonucleotides proceeds both during automated synthesis using 6-(ethoxycarbonyl)hexyl 1-O-phosphoramidite and by reaction of 5'-amino-functionalized oligonucleotides with succinic anhydride. O-(N-Succinimidyl)-1,1,3,3-tetramethyluronium tetrafluoroborate was used as activating reagent for binding of poly-L-glutamic acid to the carboxylated oligonucleotides. The successful 5'-carboxylation and poly-L-glutamic acid coupling were proven both by polyacrylamide gel electrophoreses and HPLC. 8-Aminomethylene blue in its leucoform was covalently coupled to the oligonucleotides in the presence of water soluble carbodiimide.

Base Sequence↗
PubMed2002

Chemoenzymatic synthesis and application of glycopolymers containing multivalent sialyloligosaccharides with a poly(L-glutamic acid) backbone for inhibition of infection by influenza viruses.

Highly water-soluble glycopolymers with poly(alpha-L-glutamic acid) (PGA) backbones carrying multivalent sialyl oligosaccharides units were chemoenzymatically synthesized as polymeric inhibitors of infection by human influenza viruses. p-Aminophenyl disaccharide glycosides were coupled with gamma-carboxyl groups of PGA side chains and enzymatically converted to Neu5Acalpha2-3Galbeta1-4GlcNAcbeta-, Neu5Acalpha2-6Galbeta1-4GlcNAcbeta-, Neu5Acalpha2-3Galbeta1-3GalNAcalpha-, and Neu5Acalpha2-3Galbeta1-3GalNAcbeta- units, respectively, by alpha2,3- or alpha2,6-sialytransferases. The glycopolymers synthesized were used for neutralization of human influenza A and B virus infection as assessed by measurement of the degree of cytopathic inhibitory effect in virus-infected MDCK cells. Among the glycopolymers tested, alpha2,6-sialo-PGA with a high molecular weight (260 kDa) most significantly inhibited infection by an influenza A virus, strain A/Memphis/1/71 (H3N2), which predominantly binds to alpha2-6 Neu5Ac residue. The alpha2,6-sialo-PGA also inhibited infection by an influenza B virus, B/Lee/40. The binding preference of viruses to terminal sialic acids was affected by core determinants of the sugar chain, Galbeta1-4GlcNAcbeta- or Galbeta1-3GalNAcalpha/beta- units. Inhibition of infection by viruses was remarkably enhanced by increasing the molecular weight and sialic acid content of glycopolymers.

Animals↗