Dopamine quinones interact with α-synuclein to form unstructured adducts

α-Synuclein (αsyn) fibril formation is considered a central event in the pathogenesis of Parkinson’s disease (PD). In recent years, it has been proposed that prefibrillar annular oligomeric β-sheet-rich species, called protofibrils, rather than fibrils themselves, may be the neurotoxic species. The oxidation products of dopamine (DAQ) can inhibit αsyn fibril formation supporting the idea that DAQ might stabilize αsyn protofibrils.In the present work, through different biochemical and biophysical techniques, we isolated and structurally characterized αsyn/DAQ adducts. Contrary to protofibrils, we demonstrated that αsyn/DAQ adducts retain an unfolded conformation. We then investigated the nature of the modifications induced on αsyn by DAQ. Our results indicate that only a small fraction of αsyn interacts with DAQ in a covalent way, so that non-covalent interaction appears to be the major modification induced by DAQ on αsyn.     

The conversion of the precursor form of γ-glutamyltranspeptidase to its subunit form takes place in brush border membranes

The subcellular distributions of the precursor form and mature form of γ-glutamyltranspeptidase of rat kidney were studied by labeling the enzyme with [³H] fucose in vivo. In trans Golgi elements and basolateral membranes, γ-glutamyltranspeptidase was present as a precursor form with a single polypeptide chain. However, the brush border membranes contained the heavy and light subunits as well as precursor. These results suggest that the precursor is converted to the mature form after its transport to the brush border membranes.     

In contrast to agonist monoclonal antibodies,both C-terminal truncated form and full length form of Pleiotrophin failed to activate vertebrate ALK (anaplastic lymphoma kinase)?

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase essentially and transiently expressed during development in specific regions of the central and peripheral nervous system. ALK expression persists at a lower level in the adult brain. Thus, it might play an important role in both the normal development and function of the nervous system. The nature of the cognate ligand of this receptor in vertebrates is still a matter of debate. Pleiotrophin and Midkine have been proposed as ligands of ALK but several independent studies do not confirm this hypothesis. Interestingly, a recent study proposed that a C-terminal truncated form of Pleiotrophin (Pleiotrophin.15) and not the full length form (Pleiotrophin.18) promotes Glioblastoma proliferation in an ALK-dependent fashion. These data were obviously a strong basis to conciliate the conflicting results so far reported in the literature. In the present study, we first purified to homogeneity the two forms of Pleiotrophin secreted by HEK 293 cells. In contrast to agonist monoclonal antibodies, both Pleiotrophin.15 and Pleiotrophin.18 failed to activate ALK in Neuroblastoma and Glioblastoma cells expressing this receptor. Thus, for our point of view, ALK is still an orphan receptor is vertebrates.     

Is HIV-1 evolving to a less virulent form in humans?

During the rapid spread of HIV-1 in humans, the main (M) group of HIV-1 has evolved into ten distinct subtypes, undergone countless recombination events and diversified extensively. The impact of this extreme genetic diversity on the phenotype of HIV-1 has only recently become a research focus, but early findings indicate that the dominance of HIV-1 subtype C in the current epidemic might be related to the lower virulence of this subtype compared with other subtypes. Here, we explore whether HIV-1 has reached peak virulence or has already started the slow path to attenuation.     

BAEP subcomponents and wave form — relation to click phase and stimulus rate

The latency distributions of normal brain-stem auditory evoked potential (BAEP) components elicited by condensation (C) and rarefaction (R) clicks at 10 and 50 Hz were found to be double- or multi-peaked for II (10 and 50 Hz), III (50 Hz), IV (10 and 50 Hz) and V (10 Hz). A bifid component III was found in 3.5% (10 Hz) and 7.4% (50 Hz) of BAEPs. A bifid II and triple IV/V configuration were occasionally noted. The prevalences of the different IV/V complex configurations were significantly dependent upon click phase and rate. These results suggest that several subcomponent might participate in the generation of the single BAEP components and that a single generator may contribute to different BAEP components in different subjects.Early subcomponents between I and II (Ib) were found in 13% of 10 Hz BAEPs and 27% of 50 Hz BAEPs (P = 0.002) and the latency distribution of Ib seemed to be bimodal. Ib/I relative amplitude frequently increased with 50 Hz stimulation.We suggest that the generator of Ib is partially cochlear (CM) and partially neural (equivalent to N2 of the ECochG-AP) in origin.The existence of subcomponents must be recognized in clinical use of BAEPs both to avoid misinterpretation and to decrease the normal variability in monophasic click evoked BAEP studies.     

Does the location of a mutation determine the ability to form amyloid fibrils?

We have previously reported studies of fibril formation by a set of protein G B1 domain (beta1) variants, with mutations located around the central parallel beta-strands. In this study, we designed multiple mutations in the edge strands of beta1 to create proteins with a stability range comparable to that of the set of central mutants. All the edge variants are able to form amyloid fibrils when they are incubated at their melting temperatures. This result suggests that overall protein stability is the key determinant for amyloid formation and not the specific location of destabilizing mutations. The edge strand and variants cross-seed with each other and with members of the central variant family. Interesting fibrillar morphology was observed in some cross-seeding cases and its implications for a better understanding of nucleation and elongation events are discussed.     

DNA-RNA hybrid G-quadruplex tends to form near the 3′ end of telomere overhang

Telomeric repeat-containing RNA (TERRA) has been suggested to participate in telomere maintenance. TERRA consisting of UUAGGG repeats is capable of forming an intermolecular G-quadruplex (GQ) with single-stranded TTAGGG-repeat DNA in the telomere 3′ overhang. To explore the structural features and potential functions of this DNA-RNA hybrid GQ (HGQ), we used single-molecule FRET to study the folding patterns of DNA with four to seven telomeric tandem repeats annealed with a short RNA consisting of two or five telomeric repeats. Our data highlight that RNA prefers to form DNA-RNA HGQ near the 3′ end of telomeric DNA. Furthermore, the unfolding of secondary structures by a complementary C-rich sequence was observed for DNA GQ but not for DNA-RNA HGQ, which demonstrated the enhanced stability of the telomere 3′ end via hybridization with RNA. These conformational and physical properties of telomeric DNA-RNA HGQ suggest that TERRA might limit access to the 3′ end of the telomeric DNA overhang, which is known to be critical for the interaction with telomerase and other telomere-associated proteins.     

Can copper binding to the prion protein generate a misfolded form of the protein?

The native prion protein (PrP) has a two domain structure, with a globular folded α-helical C-terminal domain and a flexible extended N-terminal region. The latter can selectively bind Cu²⁺ via four His residues in the octarepeat (OR) region, as well as two sites (His96 and His111) outside this region. In the disease state, the folded C-terminal domain of PrP undergoes a conformational change, forming amorphous aggregates high in β-sheet content. Cu²⁺ bound to the ORs can be redox active and has been shown to induce cleavage within the OR region, a process requiring conserved Trp residues. Using computational modeling, we have observed that electron transfer from Trp residues to copper can be favorable. These models also reveal that an indole-based radical cation or Cu⁺ can initiate reactions leading to protein backbone cleavage. We have also demonstrated, by molecular dynamics simulations, that Cu²⁺ binding to the His96 and His111 residues in the remaining PrP N-terminal fragment can induce localized β-sheet structure, allowing us to suggest a potential mechanism for the initiation of β-sheet misfolding in the C-terminal domain by Cu²⁺.

Can viruses form biofilms?

The recent finding that the human T-cell leukemia virus type 1 (HTLV-1) encases itself in a carbohydrate-rich adhesive extracellular 'cocoon', which enables its efficient and protected transfer between cells, unveiled a new infectious entity and a novel mechanism of viral transmission. These HTLV-1 structures are observed at the surface of T cells from HTLV-1-infected patients and are reminiscent of bacterial biofilms. The virus controls the synthesis of the matrix, which surrounds the virions and attaches them to the T cell surface. We propose that, similar to bacterial biofilms, viral biofilms could represent 'viral communities' with enhanced infectious capacity and improved spread compared with 'free' viral particles, and might constitute a key reservoir for chronic infections.     

From spatial form to system and process through pattern making in the landscape: can landscape design contribute to nature restoration?

This study presents a scenario in which recent development of landscape design based on modernism and modernists works is depicted in conjunction with its possible contribution toward nature restoration. Spatial pattern found in modernists works enhanced the designers understanding of landscape pattern underlying the land and environment at an extensive scale. In search of solid ground for pattern making, landscape designers further investigated the environmental system and its structure. A morphological approach employed in the discourse of landscape ecology gives a theoretical foundation to those endeavors. Also, the process through which the environmental system functions and is maintained becomes another potential field of design expression in landscape. Detailed studies of several works and p landscape design projects, which have already indicated a direction of development, will promote a productive and creative co-relationship between landscape design and nature restoration.This revised version was published online in April 2005 with corrections to keywords.     

Nisin Z inhibits the growth of Candida albicans and its transition from blastospore to hyphal form

Aims: To investigate the efficacy of nisin Z, an antimicrobial peptide produced by certain strains of Lactococcus lactis against Candida albicans growth and transition. Methods and Results: Candida albicans was cultured in the presence of various concentrations of nisin Z (1000, 500, and 100 μg ml⁻¹) for different time points. Candida albicans growth was determined using the Alamar Blue assay. The yeast’s transition from blastospore to hyphal form was assessed through optical microscope observations. The effect of nisin Z on C. albicans ultrastructure was followed by scanning and transmission electron microscopy. Our results show that nisin Z inhibited C. albicans growth beginning at 500 μg ml⁻¹. This inhibition was both time- and dose-dependent. Nisin Z was also active against C. albicans transition by significantly inhibiting the transformation of C. albicans from the blastospore to hyphal form. Treatments with nisin Z lead to ultrastructural disturbances of C. albicans. Conclusion: Our findings indicate that nisin Z significantly reduced C. albicans growth and transition. These effects may have occurred through ultrastructural modifications of this yeast. Significance and Impact of the Study: For the first time, effect of nisin Z on C. albicans was investigated. These results therefore suggest that nisin Z may have antifungal properties, and could be used as an antifungal molecule.     

PKA-dependent dynein switching from lysosomes to adenovirus: A novel form of host–virus competition

Cytoplasmic dynein is responsible for transport of several viruses to the nucleus. Adenovirus recruits dynein directly. Transport depends on virus-induced activation of protein kinase A (PKA) and other cellular protein kinases, whose roles in infection are poorly understood. We find that PKA phosphorylates cytoplasmic dynein at a novel site in light intermediate chain 1 (LIC1) that is essential for dynein binding to the hexon capsid subunit and for virus motility. Surprisingly, the same LIC1 modification induces a slow, but specific, dispersal of lysosomes (lyso)/late endosomes (LEs) that is mediated by inhibition of a newly identified LIC1 interaction with the RILP (Rab7-interacting lysosomal protein). These results identify an organelle-specific dynein regulatory modification that adenovirus uses for its own transport. PKA-mediated LIC1 phosphorylation causes only partial lyso/LE dispersal, suggesting a role for additional, parallel mechanisms for dynein recruitment to lyso/LEs. This arrangement provides a novel means to fine tune transport of these organelles in response to infection as well as to developmental and physiological cues.     

Disulfide-bonding between Drosophila laminin β and γ chains is essential for α chain to form αβγ trimer

Assembly of Drosophila laminin α, β and γ chains was analyzed by immunoprecipitation of the lysate from metabolically radiolabeled Kc 167 cells with chain-specific antibodies followed by two dimensional electrophoresis in which non-reducing and reducing SDS gel electrophoresis are combined. Precipitation of monomeric β (or γ) with anti-γ (or -β) antibody revealed that β and γ form stable dimer before they are disulfide-bonded to each other. In contrast, α associates with neither monomeric β, monomeric γ nor βγ dimer without disulfide-bonding but only with disulfide-bonded βγ dimer to form αβγ trimers. These results thus demonstrated that the interchain disulfide-boding between β and γ is essential for α to form αβγ trimer. We also found that the αβγ trimer can be secreted with α chain either disulfide-bonded or not bonded to the disulfide-bonded βγ dimer.     

d-Ribose glycates β(2)-microglobulin to form aggregates with high cytotoxicity through a ROS-mediated pathway

β(2)-Microglobulin (β(2)M) modified with advanced glycation end products (AGEs) is a major component of the amyloid deposits in hemodialysis-associated amyloidosis (HAA). However, the effect of glycation on the misfolding and aggregation of β(2)M has not been studied so far. Here we examine the molecular mechanism of aggregate formation of HAA-related ribosylated β(2)M in vitro. We find that the glycating agent d-ribose interacts with human β(2)M to generate AGEs that form aggregates in a time-dependent manner. Ribosylated β(2)M molecules are highly oligomerized compared with unglycated β(2)M, and have granular morphology. Furthermore, such ribosylated β(2)M aggregates show significant cytotoxicity to both human SH-SY5Y neuroblastoma and human foreskin fibroblast FS2 cells and induce intracellular reactive oxygen species (ROS). Presence of the antioxidant N-acetylcysteine (1.0mM) attenuated intracellular ROS and prevented cell death induction in both SH-SY5Y and FS2 cells, indicating that the cytotoxicity of ribosylated β(2)M aggregates depends on a ROS-mediated pathway in both cell lines. In other words, d-ribose reacts with β(2)M and induces the ribosylated protein to form granular aggregates with high cytotoxicity through a ROS-mediated pathway. These findings suggest that ribosylated β(2)M aggregates could contribute to the dysfunction and death of cells and could play an important role in the pathogenesis of β(2)M-associated diseases such as HAA.     

Should you form a group?

In this discussion, greatest emphasis has been placed upon the personal factors involved, rather than upon the mechanical aspects of creating and maintaining a group, since it is the personal factors, the authors say, that are the most often overlooked.     

The ability to convert the 4 S glucocorticoid receptor to the 7–8 S form is dependent on both RNA and protein factors

The DEAE-cellulose-purified 4 S form of the rat liver glucocorticoid receptor can associate with cytosolic factors, as evidenced by an alteration of the sedimentation value of the 7–8 S form. On the basis of sedimentation profile, this form is indistinguishable from the activated, low-salt 7–8 S form isolated from rat liver cytosol. In addition, both the endogenous and reconstituted 7–8 S receptor can bind DNA as the 7–8 S form. In keeping with our reports that the endogenous form of the 7–8 S receptor is sensitive to RNAase digestion, treatment of the cytosol with RNAase prior to mixing with the 4 S receptor prevents the formation of the 7–8 S material. Moreover, warming the cytosol to 50°C prior to mixing with the 4 S receptor also eliminates the ability to form the heavier material. Since RNA is heat-stable, this suggests that other factors may be involved. Treatment of the cytosol with N-ethylmaleimide, a reagent reported to be specific for sulfhydryl groups, also eliminates 7–8 S generating ability. These observations suggest that a protein may be a component of the 7–8 S generating material. This is substantiated by the observation that trypsin or chymotrypsin treatment of the cytosol mitigates the ability of the cytosol to form the 7–8 S material and results in the appearance of a form of the receptor that sediments at approximately 6 S. Protease treatment of partially purified material eliminates the 7–8 S generating activity entirely. We conclude that the 7–8 S form of the receptor can be reconstituted from the 4 S receptor via association with at least two other cytosolic factors, a protein and an RNA.     

The α1 and α2 domains of H-2 class I molecules interact to form unique epitopes

Mouse class I antigens are the major targets of cytolytic T lymphocytes in both major histocompatibility complex (MHC)-restricted and allogeneic responses. Considerable evidence has recently accumulated demonstrating that MHC class I molecules encoded by genes whose 1 and 2 coding exons were interchanged are not recognized by T lymphocytes specific for parental class I products. Along with the loss of T -cell reactivity, there is a loss of recognition by some, but not all monoclonal antibodies. In this communication we report that the loss of reactivity by monoclonal antibodies is accompanied by the gain of new epitopes caused by the interaction of 1 and 2 domains. These epitopes are immunodominant. They are the major determinant recognized by polyclonal antisera raised by immunization with L cells transfected with exon-shuffled class I genes. Four new monoclonal antibodies have been produced which recognize at least two separate epitopes caused by the interaction of the 1^P and 2^d domains.     

Zn2+-Aβ40 complexes form metastable quasi-spherical oligomers that are cytotoxic to cultured hippocampal neurons

The roles of metal ions in promoting amyloid β-protein (Aβ) oligomerization associated with Alzheimer disease are increasingly recognized. However, the detailed structures dictating toxicity remain elusive for Aβ oligomers stabilized by metal ions. Here, we show that small Zn(2+)-bound Aβ1-40 (Zn(2+)-Aβ40) oligomers formed in cell culture medium exhibit quasi-spherical structures similar to native amylospheroids isolated recently from Alzheimer disease patients. These quasi-spherical Zn(2+)-Aβ40 oligomers irreversibly inhibit spontaneous neuronal activity and cause massive cell death in primary hippocampal neurons. Spectroscopic and x-ray diffraction structural analyses indicate that despite their non-fibrillar morphology, the metastable Zn(2+)-Aβ40 oligomers are rich in β-sheet and cross-β structures. Thus, Zn(2+) promotes Aβ40 neurotoxicity by structural organization mechanisms mediated by coordination chemistry.