Nano-plastics in the aquatic environment.

The amount of plastics released to the environment in modern days has increased substantially since the development of modern plastics in the early 1900s. As a result, concerns have been raised by the public about the impact of plastics on nature and on, specifically, aquatic wildlife. Lately, much attention has been paid to macro- and micro-sized plastics and their impact on aquatic organisms. However, micro-sized plastics degrade subsequently into nano-sizes whereas nano-sized particles may be released directly into nature. Such particles have a different impact on aquatic organisms than larger pieces of plastic due to their small size, high surface curvature, and large surface area. This review describes the possible sources of nano-sized plastic, its distribution and behavior in nature, the impact of nano-sized plastic on the well-being of aquatic organisms, and the difference of impact between nano- and micro-sized particles. We also identify research areas which urgently need more attention and suggest experimental methods to obtain useful data.

Phosphorylation of the Saccharomyces cerevisiae La protein does not appear to be required for its functions in tRNA maturation and nascent RNA stabilization.

An abundant nuclear phosphoprotein, the La autoantigen, is the first protein to bind all newly synthesized RNA polymerase III transcripts. Binding by the La protein to the 3' ends of these RNAs stabilizes the nascent transcripts from exonucleolytic degradation. In the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, the La protein is required for the normal pathway of tRNA maturation. Experiments in which the human protein was expressed in S. pombe have suggested that phosphorylation of the La protein regulates tRNA maturation. To dissect the role of phosphorylation in La protein function, we used mass spectrometry to identify three sites of serine phosphorylation in the S. cerevisiae La protein Lhp1p. Mutant versions of Lhp1p, in which each of the serines was mutated to alanine, were expressed in yeast cells lacking Lhp1p. Using two-dimensional gel electrophoresis, we determined that we had identified and mutated all major sites of phosphorylation in Lhp1p. Lhp1p lacking all three phosphorylation sites was functional in several yeast strains that require Lhp1p for growth. Northern blotting revealed no effects of Lhp1p phosphorylation status on either pre-tRNA maturation or stabilization of nascent RNAs. Both wild-type and mutant Lhp1 proteins localized to both nucleoplasm and nucleoli, demonstrating that phosphorylation does not affect subcellular location. Thus, although La proteins from yeast to humans are phosphoproteins, phosphorylation does not appear to be required for any of the identified functions of the S. cerevisiae protein.

Carbohydrate groups of alpha1-microglobulin are important for secretion and tissue localization but not for immunological properties.

The role of the carbohydrates for the structure and functions of the plasma and tissue protein alpha1-microglobulin (alpha1m) was investigated by deletion of the sites for N-glycosylation by site-directed mutagenesis (N17,96-->Q). The mutated cDNA was expressed in a baculovirus-insect cell system resulting in a nonglycosylated protein. The biochemical properties of N17,96Q-alpha1m were compared to nonmutated alpha1m, which carries two short non-sialylated N-linked oligosaccharides when expressed in the same system. Both proteins carried a yellow-brown chromophore and were heterogeneous in charge. Circular dichroism spectra and antibody binding indicated a similar overall structure. However, the secretion of N17,96Q-alpha1m was significantly reduced and approximately 75% of the protein were found accumulated intracellularly. The in vitro immunological effects of recombinant nonmutated alpha1m and N17,96Q-alpha1m were compared to the effects of alpha1m isolated from plasma, which is sialylated and carries an additional O-linked oligosaccharide. All three alpha1m variants bound to human peripheral lymphocytes and mouse T cell hybridomas to the same extent. They also inhibited the antigen-stimulated proliferation of peripheral lymphocytes and antigen-stimulated interleukin 2-secretion of T cell hybridomas in a similar manner. After injection of rats intravenously, the blood clearance of recombinant nonmutated and N17,96Q-alpha1m was faster than that of plasma alpha1m. Nonmutated alpha1m was located primarily to the liver, most likely via binding to asialoglycoprotein receptors, and N17,96Q-alpha1m was located mainly to the kidneys. It is concluded that the carbohydrates of alpha1m are important for the secretion and the in vivo turnover of the protein, but not for the structure or immunological properties.

Alpha1-microglobulin chromophores are located to three lysine residues semiburied in the lipocalin pocket and associated with a novel lipophilic compound.

Alpha1-microglobulin (alpha1m) is an electrophoretically heterogeneous plasma protein. It belongs to the lipocalin superfamily, a group of proteins with a three-dimensional (3D) structure that forms an internal hydrophobic ligand-binding pocket. Alpha1m carries a covalently linked unidentified chromophore that gives the protein a characteristic brown color and extremely heterogeneous optical properties. Twenty-one different colored tryptic peptides corresponding to residues 88-94, 118-121, and 122-134 of human alpha1m were purified. In these peptides, the side chains of Lys92, Lys118, and Lys130 carried size heterogeneous, covalently attached, unidentified chromophores with molecular masses between 122 and 282 atomic mass units (amu). In addition, a previously unknown uncolored lipophilic 282 amu compound was found strongly, but noncovalently associated with the colored peptides. Uncolored tryptic peptides containing the same Lys residues were also purified. These peptides did not carry any additional mass (i.e., chromophore) suggesting that only a fraction of the Lys92, Lys118, and Lys130 are modified. The results can explain the size, charge, and optical heterogeneity of alpha1m. A 3D model of alpha1m, based on the structure of rat epididymal retinoic acid-binding protein (ERABP), suggests that Lys92, Lys118, and Lys130 are semiburied near the entrance of the lipocalin pocket. This was supported by the fluorescence spectra of alpha1m under native and denatured conditions, which indicated that the chromophores are buried, or semiburied, in the interior of the protein. In human plasma, approximately 50% of alpha1m is complex bound to IgA. Only the free alpha1m carried colored groups, whereas alpha1m linked to IgA was uncolored.

Coiled-coil structure of group A streptococcal M proteins. Different temperature stability of class A and C proteins by hydrophobic-nonhydrophobic amino acid substitutions at heptad positions a and d.

M proteins and M-like proteins, expressed on the surface of group A streptococci and binding to human plasma proteins, can be divided into two classes, A and C, depending on the structure of the central repeated regions. The class C proteins have been shown to be dimers with a coiled-coil structure. In this work, we have compared the structure and binding of a class A protein, Mrp4, and a class C protein, Arp4, expressed by the same bacterial strain. Circular dichroism spectra, gel filtration, and binding assays showed that both proteins had a coiled-coil dimer configuration and a high-affinity binding at 20 degrees C. However, striking differences were seen at 37 degrees C. The class A protein, Mrp4, was still a coiled-coil dimer with high affinity binding activity, whereas the class C protein, Arp4, had lost both the coiled-coil structure and binding activity. Raising the temperature even higher, Mrp4 retained the coiled-coil structure up to 70-90 degrees C. Furthermore, a recombinant protein, Mrp(C), in which the A-repeats of Mrp4 were replaced by the C-repeats of Arp4, lost its coiled-coil structure and fibrinogen-binding around 40-45 degrees C. These results suggest a high thermal stability of class A proteins and a low stability of class C proteins and that the structural basis for this can be found partly in the A- and C-repeats. Analysis of the amino acid sequences of the A- and C-repeats, revealed a large difference, 87% and 45%, respectively, in the content of hydrophobic amino acid residues in the positions regarded as important for the formation of the coiled-coil structure. In particular, several alanine residues in the A-repeats were replaced by serine residues in the C-repeats. Our results suggest that important structural and functional changes within the M protein family have evolved by specific hydrophobic-nonhydrophobic amino acid replacements.

Allosteric and temperature effects on the plasma protein binding by streptococcal M protein family members.

Most group A streptococcal strains bind immunoglobulins (Ig) and fibrinogen to their cell walls. It is shown in this paper that the Ig-binding of three different strains was much weaker at 37 degrees C than at room temperature (20 degrees C), whereas the fibrinogen binding was unaffected by temperature. The binding properties and molecular sizes of two purified group A streptococcal cell surface proteins from the M protein family were studied at various temperatures, M1 protein with affinity for IgG, fibrinogen and albumin, and protein Sir22 with affinity for IgA and IgG. Both proteins appeared as monomers which bound all their ligands, including fibrinogen, very weakly at 37 degrees C, and as strongly binding dimers at 10 and 20 degrees C. Furthermore, the results demonstrated that the plasma protein binding of the bacterial proteins was allosterically regulated, i.e. the binding of a ligand to one site modulated the binding of a ligand to a second site. For example, the binding of albumin or IgG to purified M1 protein at 10 and 20 degrees C strongly enhanced the binding of fibrinogen at 37 degrees C. This indicates that the high affinity dimer form of the bacterial proteins can be stabilized at 37 degrees C, a possible explanation for the strong fibrinogen binding of whole bacteria. Finally, the sizes and binding properties of three M1 protein fragments were studied and the results indicated that the centrally located C-repeats, which are conserved among the members of the M protein family, are important for the formation of the high-affinity dimers of the bacterial proteins.

Processing and secretion of rat alpha 1-microglobulin-bikunin expressed in eukaryotic cell lines.

The precursor protein alpha 1-microglobulin-bikunin was cleaved to the same degree whether expressed in CHO cells or in mutated CHO cells, RPE.40 cells, suggested to lack a functional form of the intracellular protease furin. Thus, alpha 1-microglobulin-bikunin probably is not cleaved in vivo by furin. However, simultaneous overexpression of the precursor and furin in COS, CHO and RPE.40 cells increased the cleavage, suggesting that compartmentalisation and concentrations of protease and precursor are important for the cleavage, besides the in vitro specificity. Expression of alpha 1-microglobulin and bikunin alone gave different protein patterns of SDS-PAGE as compared to expression of the precursor and subsequent cleavage, suggesting that the precursor protein is important for the post-translational handling of alpha 1-microglobulin and bikunin.