Copper in disorders with neurological symptoms: Alzheimer`s, Menkes, and Wilson Diseases

Copper is an essential element for the activity of a number of physiologically important enzymes. Enzyme-related malfunctions may contribute to severe neurological symptoms and neurological diseases: copper is a component of cytochrome c oxidase, which catalyzes the reduction of oxygen to water, the essential step in cellular respiration. Copper is a cofactor of Cu/Zn-superoxide-dismutase which plays a key role in the cellular response to oxidative stress by scavenging reactive oxygen species. Furthermore, copper is a constituent of dopamine-β-hydroxylase, a critical enzyme in the catecholamine biosynthetic pathway. A detailed exploration of the biological importance and functional properties of proteins associated with neurological symptoms will have an important impact on understanding disease mechanisms and may accelerate development and testing of new therapeutic approaches. Copper binding proteins play important roles in the establishment and maintenance of metal-ion homeostasis, in deficiency disorders with neurological symptoms (Menkes disease, Wilson disease) and in neurodegenerative diseases (Alzheimer’s disease). The Menkes and Wilson proteins have been characterized as copper transporters and the amyloid precursor protein (APP) of Alzheimer’s disease has been proposed to work as a Cu(II) and/or Zn(II) transporter. Experimental, clinical and epidemiological observations in neurodegenerative disorders like Alzheimer’s disease and in the genetically inherited copper-dependent disorders Menkes and Wilson disease are summarized. This could provide a rationale for a link between severely dysregulated metal-ion homeostasis and the selective neuronal pathology.

Insights into exazaphosphorine resistance and possible approaches to its circumvention

The oxazaphosphorines cyclophosphamide, ifosfamide and trofosfamide remain a clinically useful class of anticancer drugs with substantial antitumour activity against a variety of solid tumors and hematological malignancies. A major limitation to their use is tumour resistance, which is due to multiple mechanisms that include increased DNA repair, increased cellular thiol levels, glutathione S-transferase and aldehyde dehydrogenase activities, and altered cell-death response to DNA damage. These mechanisms have been recently re-examined with the aid of sensitive analytical techniques, high-throughput proteomic and genomic approaches, and powerful pharmacogenetic tools. Oxazaphosphorine resistance, together with dose-limiting toxicity (mainly neutropenia and neurotoxicity), significantly hinders chemotherapy in patients, and hence, there is compelling need to find ways to overcome it. Four major approaches are currently being explored in preclinical models, some also in patients: combination with agents that modulate cellular response and disposition of oxazaphosphorines; antisense oligonucleotides directed against specific target genes; introduction of an activating gene (CYP3A4) into tumor tissue; and modification of dosing regimens. Of these approaches, antisense oligonucleotides and gene therapy are perhaps more speculative, requiring detailed safety and efficacy studies in preclinical models and in patients. A fifth approach is the design of novel oxazaphosphorines that have favourable pharmacokinetic and pharmacodynamic properties and are less vulnerable to resistance. Oxazaphosphorines not requiring hepatic CYP-mediated activation (for example, NSC 613060 and mafosfamide) or having additional targets (for example, glufosfamide that also targets glucose transport) have been synthesized and are being evaluated for safety and efficacy. Characterization of the molecular targets associated with oxazaphosphorine resistance may lead to a deeper understanding of the factors critical to the optimal use of these agents in chemotherapy and may allow the development of strategies to overcome resistance.

Global and targeted gene expression and protein content in skeletal muscle of young men following short-term creatine monohydrate supplementation

Creatine monohydrate (CrM) supplementation has been shown to increase fat-free mass and muscle power output possibly via cell swelling. Little is known about the cellular response to CrM. We investigated the effect of short-term CrM supplementation on global and targeted mRNA expression and protein content in human skeletal muscle. In a randomized, placebo-controlled, crossover, double-blind design, 12 young, healthy, nonobese men were supplemented with either a placebo (PL) or CrM (loading phase, 20 g/day x 3 days; maintenance phase, 5 g/day x 7 days) for 10 days. Following a 28-day washout period, subjects were put on the alternate supplementation for 10 days. Muscle biopsies of the vastus lateralis were obtained and were assessed for mRNA expression (cDNA microarrays + real-time PCR) and protein content (Kinetworks KPKS 1.0 Protein Kinase screen). CrM supplementation significantly increased fat-free mass, total body water, and body weight of the participants (P < 0.05). Also, CrM supplementation significantly upregulated (1.3- to 5.0-fold) the mRNA content of genes and protein content of kinases involved in osmosensing and signal transduction, cytoskeleton remodeling, protein and glycogen synthesis regulation, satellite cell proliferation and differentiation, DNA replication and repair, RNA transcription control, and cell survival. We are the first to report this large-scale gene expression in the skeletal muscle with short-term CrM supplementation, a response that suggests changes in cellular osmolarity.

Repair and tolerance of DNA damage in higher plants

DNA repair mechanisms constitute an essential cellular response to DNA damage arising either from metabolic processes or from environmental sources such as ultraviolet radiation. Repair of these lesions may be via direct reversal, or by processes such as nucleotide excision repair (NER), a coordinated pathway in which lesions and the surrounding nucleotides are excised and replaced via DNA resynthesis. The importance of repair is illustrated by human disease states such as xeroderma pigmentosum and Cockayne's syndrome which result from defects in the NER system arising from mutations in XP- genes or XP- and CS- genes respectively Little detail is known of DNA damage repair processes in plants, despite the economic and ecological importance of these organisms. This study aimed to expand our knowledge of the process of NER in plants, largely via a polymerase chain reaction (PCR)-based approach involving amplification, cloning and characterisation of plant genomic DNA and cDNA. Homologues of the NER components XPF/RAD1 and XPD/RAD3 were isolated as both genomic and complete cDNA sequences from the model dicotyledonous plant Arabidopsis thaliana. The sequence of the 3'-untranslated region of atXPD was also determined. Comparison of genomic and cDNA sequences allowed a detailed analysis of gene structures, including details of intron/exon processing. Variable transcript processing to produce three distinct transcripts was found in the case of atXPF. In an attempt to validate the proposed homologous function of these cDNAs, assays to test complementation of resistance to ultraviolet radiation in the relevant yeast mutants were performed. Despite extensive amino acid sequence conservation, neither plant cDNA was able to restore UV-resistance. As the yeast RAD3 gene product is also involved in vivo in transcription, and so is required for viability, the atXPD cDNA was tested in a complementation assay for this function in an appropriate yeast mutant. The plant cDNA was found to substantially increase the viability of the yeast mutant. The structural and functional significance of these results is discussed comparatively with reference to yeast, human and other known homologues. Other putative NER homologues were identified in A. thaliana database sequences, including those of ERCC1/RAD10 and XPG/ERCC5/RAD2, and are now the subjects of ongoing investigations. This study also describes preliminary investigations of putative REVS and RAD30 translesion synthesis genes from A. thaliana.

Natriuretic peptide receptor signalling in the gills of fishes

Natriuretic peptide hormones exert their effects by binding to receptor proteins and activating cellular responses. Fish gills interface with the aquatic environment. Therefore, natriuretic peptide receptor activity in gills from marine and freshwater fishes was examined. The potencies of natriuretic peptides were compared and a novel cellular response was identified.

The ZntA-like NpunR4017 plays a key role in maintaining homeostatic levels of zinc in Nostoc punctiforme

Analysis of cellular response to zinc exposure provides insights into how organisms maintain homeostatic levels of zinc that are essential, while avoiding potentially toxic cytosolic levels. Using the cyanobacterium Nostoc punctiforme as a model, qRT-PCR analyses established a profile of the changes in relative mRNA levels of the ZntA-like zinc efflux transporter NpunR4017 in response to extracellular zinc. In cells treated with 18 μM of zinc for 1 h, NpunR4017 mRNA levels increased by up to 1300 % above basal levels. The accumulation and retention of radiolabelled (65)Zn by NpunR4107-deficient and overexpressing strains were compared to wild-type levels. Disruption of NpunR4017 resulted in a significant increase in zinc accumulation up to 24 % greater than the wild type, while cells overexpressing NpunR4107 accumulated 22 % less than the wild type. Accumulation of (65)Zn in ZntA(-) Escherichia coli overexpressing NpunR4017 was reduced by up to 21 %, indicating the capacity for NpunR4017 to compensate for the loss of ZntA. These findings establish the newly identified NpunR4017 as a zinc efflux transporter and a key transporter for maintaining zinc homeostasis in N. punctiforme.

Modulated deformation of lipid membrane to vesicles and tubes due to reduction of graphene oxide substrate under laser irradiation

Biomembrane transformations are closely related to many biological processes including endo/exocytosis and the cellular response to the local physical environment. In this work, we investigated the transformation between lipid membranes and lipid vesicles/tubes modulated by the solid substrate of graphene oxide (GO) aggregates under laser irradiation. We firstly fabricate a novel type of lipid@GO composite consisting of micrometer-sized GO aggregates surrounded by lamellar lipid membranes. Upon laser irradiation, lipid protrusion occurs and leads to the formation of vesicles adsorbed on the GO aggregate surface, with an average size as 0.43 times of the radius of GO aggregate. Both the location and the dynamic formation process of vesicles can be modulated. The arising of vesicles prefers to occur at edges of the GO planes rather than on surface of individual GO sheets within the GO aggregate. Furthermore, at a reduced laser power density, the lipid protrusion mainly grows to tubes instead of vesicles. Such transformations from lipid membrane to vesicles and tubes is ascribed to the reduction of GO to reduced-GO (rGO) under laser irradiation, probably along with the release of gases leading to the deformation of lipid membrane surrounding the GO surface.

Sex-related differences in the organismal and cellular stress response in juvenile salmon exposed to treated bleached kraft mill effluent

Exposure of fish to stressors can elicit biochemical and organismal changes at multiple levels of biological organization collectively known as stress responses. The organismal (plasma glucose and cortisol levels) and cellular (hepatic hsp70) stress responses in fish have been studied in several species, but little is known about sex-related differences in these responses. In this study, we exposed sexually immature juvenile chinook salmon (Oncorhynchus tshawytscha) to bleached kraft mill effluent (BKME: 0%, 1%, and 10% v/v) for 30 days and then measured components of their organismal and cellular stress responses. Males exposed to 1% BKME had higher levels of plasma glucose than females. Plasma cortisol levels were unaffected in females exposed to BKME, but males exposed to 10% BKME had significantly higher levels of plasma cortisol relative to non-exposed males. While exposure to BKME did not affect hsp70 levels in males, females exposed to 1% BKME had higher levels of hsp70 relative to non-exposed and 10% BKME groups. Within any given treatment, females had higher levels of hsp70 relative to males. This study demonstrates that sex-related differences exist in commonly used indicators of stress in fish, and points out the importance of considering the sex of the fish in stress research.

Expression of Na+/H+ exchanger mRNA in the gills of the Atlantic hagfish (Myxine glutinosa) in response to metabolic acidosis

Sodium/proton exchangers (NHE) are transmembrane proteins that facilitate the exchange of a Na⁺ ion for a H⁺ ion across cellular membranes. The NHE are present in the gills of fishes and are believed to function in acid-base regulation by driving the extrusion of protons across the branchial epithelium in exchange for Na⁺ in the water. In this study, we have used reverse transcriptase-polymerase chain reaction (RT-PCR) to detect the presence of a branchial NHE in the gills of the Atlantic hagfish, Myxine glutinosa. The subsequent partial cDNA sequence shares homology with other vertebrate and invertebrate NHE isoforms. In addition, using semi-quantitative, multiplex RT-PCR we demonstrate that mRNA expression of hagfish gill NHE is upregulated following an induced metabolic acidosis. Expression was increased to 4.4 times basal levels at 2-h post-infusion and had decreased to 1.6 times basal by 6 h. Expression had returned to basal levels by 24-h post-infusion. The inference from this study is that a gill NHE which is potentially important in acid-base regulation has been present in the vertebrate lineage since before the divergence of the hagfishes from the main vertebrate line.

Potassium phosphonate alters the defence response of Xanthorrhoea australis following infection by Phytophthora cinnamomi

Potassium phosphonate (phosphite) is widely used in the management of Phytophthora diseases in agriculture, horticulture and natural environments. The Austral grass tree, Xanthorrhoea australis, a keystone species in the dry sclerophyll forests of southern Australia, is susceptible to Phytophthora cinnamomi, but is protected by applications of phosphite. We examined the effect of phosphite application on the infection of X. australis seedlings and cell suspension cultures by zoospores of P. cinnamomi. Phosphite induced more intense cellular responses to pathogen challenge and suppressed pathogen ingress in both seedlings and cell cultures. In untreated X. australis seedlings, hyphal growth was initially intercellular, became intracellular 24 h after inoculation, and by 48 h had progressed into the vascular tissue. In phosphite-treated seedlings, growth of P. cinnamomi remained intercellular and was limited to the cortex, even at 72 h after inoculation. The cell membrane retracted from the cell wall and phenolic compounds and electron dense substances were deposited around the wall of infected and neighbouring cells. Suspension cells were infected within 6 h of inoculation. Within 24 h of inoculation, untreated cells were fully colonised, had collapsed cytoplasm and died. The protoplast of phosphite-treated suspension cells collapsed within 12 h of inoculation, and phenolic material accumulated in adjacent, uninfected cells. No anatomical response to phosphite treatment was observed before infection of plant tissues, suggesting that the phosphite-associated host defence response is induced following pathogen challenge. Anatomical changes provide evidence that phosphite stimulates the host defence system to respond more effectively to pathogen invasion.

Cellular adaption to repeated eccentric exercise-induced muscle damage

Eccentrically biased exercise results in skeletal muscle damage and stimulates adaptations in muscle, whereby indexes of damage are attenuated when the exercise is repeated. We hypothesized that changes in ultrastructural damage, inflammatory cell infiltration, and markers of proteolysis in skeletal muscle would come about as a result of repeated eccentric exercise and that gender may affect this adaptive response. Untrained male (n = 8) and female (n = 8) subjects performed two bouts (bout 1 and bout 2), separated by 5.5 wk, of 36 repetitions of unilateral, eccentric leg press and 100 repetitions of unilateral, eccentric knee extension exercises (at 120% of their concentric single repetition maximum), the subjects' contralateral nonexercised leg served as a control (rest). Biopsies were taken from the vastus lateralis from each leg 24 h postexercise. After bout 2, the postexercise force deficit and the rise in serum creatine kinase (CK) activity were attenuated. Women had lower serum CK activity compared with men at all times (P < 0.05), but there were no gender differences in the relative magnitude of the force deficit. Muscle Z-disk streaming, quantified by using light microscopy, was elevated vs. rest only after bout 1 (P < 0.05), with no gender difference. Muscle neutrophil counts were significantly greater in women 24 h after bout 2 vs. rest and bout 1 (P < 0.05) but were unchanged in men. Muscle macrophages were elevated in men and women after bout 1 andbout 2 (P < 0.05). Muscle protein content of the regulatory calpain subunit remained unchanged whereas ubiquitin-conjugated protein content was increased after both bouts (P < 0.05), with a greater increase after bout 2. We conclude that adaptations to eccentric exercise are associated with attenuated serum CK activity and, potentially, an increase in the activity of the ubiquitin proteosome proteolytic pathway.

Diverse fibrillar peptides directly bind the Alzheimer’s amyloid precursor protein and amyloid precursor-like protein 2 resulting in cellular accumulation

The Alzheimer’s disease Aβ peptide can increase the levels of cell-associated amyloid precursor protein (APP) in vitro. To determine the specificity of this response for Aβ and whether it is related to cytotoxicity, we tested a diverse range of fibrillar peptides including amyloid-β (Aβ), the fibrillar prion peptides PrP106–126 and PrP178–193 and human islet-cell amylin. All these peptides increased the levels of APP and amyloid precursor-like protein 2 (APLP2) in primary cultures of astrocytes and neurons. Specificity was shown by a lack of change to amyloid precursor-like protein 1, τ-1 and cellular prion protein (PrPc) levels. APP and APLP2 levels were elevated only in cultures exposed to fibrillar peptides as assessed by electron microscopy and not in cultures treated with non-fibrillogenic peptide variants or aggregated lipoprotein. We found that PrP106–126 and the non-toxic but fibril-forming PrP178–193 increased APP levels in cultures derived from both wild-type and PrPc-deficient mice indicating that fibrillar peptides up-regulate APP through a non-cytotoxic mechanism and irrespective of parental protein expression. Fibrillar PrP106–126 and Aβ peptides bound recombinant APP and APLP2 suggesting the accumulation of these proteins was mediated by direct binding to the fibrillated peptide. This was supported by decreased APP accumulation following extensive washing of the cultures to remove fibrillar aggregates. Pre-incubation of fibrillar peptide with recombinant APP18–146, the putative fibril binding site, also abrogated the accumulation of APP. These findings show that diverse fibrillogenic peptides can induce accumulation of APP and APLP2 and this mechanism could contribute to pathogenesis in neurodegenerative disorders.

Protection and compensation in the influenza virus-specific CD8+ T cell response.

Influenza virus-specific CD8+ T cells generally recognize peptides derived from conserved, internal proteins that are not subject to antibody-mediated selection pressure. Prior exposure to any one influenza A virus (H1N1) can prime for a secondary CD8+ T cell response to a serologically different influenza A virus (H3N2). The protection afforded by this recall of established CD8+ T cell memory, although limited, is not negligible. Key characteristics of primary and secondary influenza-specific host responses are probed here with recombinant viruses expressing modified nucleoprotein (NP) and acid polymerase (PA) genes. Point mutations were introduced into the epitopes derived from the NP and PA such that they no longer bound the presenting H2Db MHC class I glycoprotein, and reassortant H1N1 and H3N2 viruses were made by reverse genetics. Conventional (C57BL/6J, H2b, and Ig+/+) and Ig-/- (muMT) mice were more susceptible to challenge with the single NP [HKx31 influenza A virus (HK)-NP] and PA (HK-PA) mutants, but unlike the Ig-/- mice, Ig+/+ mice were surprisingly resistant to the HK-NP/-PA double mutant. This virus was found to promote an enhanced IgG response resulting, perhaps, from the delayed elimination of antigen-presenting cells. Antigen persistence also could explain the increase in size of the minor KbPB1703 CD8+ T cell population in mice infected with the mutant viruses. The extent of such compensation was always partial, giving the impression that any virus-specific CD8+ T cell response operates within constrained limits. It seems that the relationship between protective humoral and cellular immunity is neither simple nor readily predicted.