Iron dysregulation in Alzheimer's disease: multimodal brain permeable iron chelating drugs, possessing neuroprotective-neurorescue and amyloid precursor protein-processing regulatory activities as therapeutic agents

Considering the multi-etiological character of Alzheimer's disease (AD), the current pharmacological approaches using drugs oriented towards a single molecular target possess limited ability to modify the course of the disease and thus, offer a partial benefit to the patient. In line with this concept, novel strategies include the use of a cocktail of several drugs and/or the development of a single molecule, possessing two or more active neuroprotective-neurorescue moieties that simultaneously manipulate multiple targets involved in AD pathology. A consistent observation in AD is a dysregulation of metal ions (Fe²⁺, Cu²⁺ and Zn²⁺) homeostasis and consequential induction of oxidative stress, associated with beta-amyloid aggregation and neurite plaque formation. In particular, iron has been demonstrated to modulate the Alzheimer's amyloid precursor holo-protein expression by a pathway similar to that of ferritin L-and H-mRNA translation through iron-responsive elements in their 5′UTRs. This review will discuss two separate scenarios concerning multiple therapy targets in AD, sharing in common the implementation of iron chelation activity: (i) novel multimodal brain-permeable iron chelating drugs, possessing neuroprotective-neurorescue and amyloid precursor protein-processing regulatory activities; (ii) natural plant polyphenols (flavonoids), such as green tea epigallocatechin gallate (EGCG) and curcumin, reported to have access to the brain and to possess multifunctional activities, such as metal chelation-radical scavenging, anti-inflammation and neuroprotection.     

Rhythm perception: Speeding up or slowing down affects different subcomponents of the ERP P3 complex

The aim of this study was to investigate, by measuring the event related potential (ERP) P3 complex, whether the perception of small accelerations differs from that of small decelerations. Participants had to decide whether the last beat of a short sequence was presented 'too early' or 'too late'. Target beats were accelerated or decelerated with 0%, 2%, 5%, or 10%. Individuals differed in their capability to detect small tempo changes. We found that good responders were able to identify all tempo changes whereas poor responders were only able to identify large (10%) tempo changes. In addition, we found that tempo changes affected two subcomponents of the ERP P3 in good performers. Accelerations increased a late-P3 amplitude whereas decelerations increased an early-P3 amplitude. These results imply the principle possibility to measure differential P3 effects within one task. This is important for acquiring more refined knowledge concerning different subcomponents of the ERP P3 complex and the cognitive processes by which they are elicited.     

Transient expression of thyroid hormone nuclear receptor TRbeta2 sets S opsin patterning during cone photoreceptor genesis.

Cone photoreceptors in the murine retina are patterned by dorsal repression and ventral activation of S opsin. TR beta 2, the nuclear thyroid hormone receptor beta isoform 2, regulates dorsal repression. To determine the molecular mechanism by which TR beta 2 acts, we compared the spatiotemporal expression of TR beta 2 and S opsin from embryonic day (E) 13 through adulthood in C57BL/6 retinae. TR beta 2 and S opsin are expressed in cone photoreceptors only. Both are transcribed by E13, and their levels increase with cone genesis. TR beta 2 is expressed uniformly, but transiently, across the retina. mRNA levels are maximal by E17 at completion of cone genesis and again minimal before P5. S opsin is also transcribed by E13, but only in ventral cones. Repression in dorsal cones is established by E17, consistent with the occurrence of patterning during cone cell genesis. The uniform expression of TR beta 2 suggests that repression of S opsin requires other dorsal-specific factors in addition to TR beta 2. The mechanism by which TR beta 2 functions was probed in transgenic animals with TR beta 2 ablated, TR beta 2 that is DNA binding defective, and TR beta 2 that is ligand binding defective. These studies show that TR beta 2 is necessary for dorsal repression, but not ventral activation of S opsin. TR beta 2 must bind DNA and the ligand T3 (thyroid hormone) to repress S opsin. Once repression is established, T3 no longer regulates dorsal S opsin repression in adult animals. The transient, embryonic action of TR beta 2 is consistent with a role (direct and/or indirect) in chromatin remodeling that leads to permanent gene silencing in terminally differentiated, dorsal cone photoreceptors.     

人趋化因子CCL3L1融合蛋白表达和活性分析

目的人趋化因子CCL3L1进行融合蛋白原核表达和真核表达，纯化后活性分析。方法克隆人类CCL3L1eDNA，构建两种CCL3L1表达载体，获得两个CCL3L1融合蛋白，一个在BL21大肠杆菌表达的GST-CCL3L1融合蛋白，另一个在跎果蝇细胞表达的His-CCL3L1融合蛋白。同时克隆了pcDNA3.1-flag-CCR5表达载体，培养了稳定表达flag-CCR5的细胞株，进行人趋化因子CCL3L1活性分析。结果成功构建人趋化因子CCL3L1融合蛋白原核表达载体pGEX-4T和真核表达载体pMT／BiP／V5-His，免疫沉淀法检测和Westernblot法分析His-CCL3L1蛋白在浓度1nmol／L到50nmol／L存在剂量依赖性，浓度50nmol／L到100nmol／L没有剂量依赖性。纯化的His-CCL3L1蛋白能特异性结合CCR5受体。结论成功表达了融合蛋白GST-CCL3L1和His-CCL3LI，果蝇细胞表达的His-CCL3L1蛋白具有与天然CCL3L1相同的生物学活性，为进一步制备CCL3L1单克隆和多克隆抗体及研究CCL3L1影响HIV-1感染的机制提供基础资料。     

Preparation and Fluorescent and Magnetic Properties of Polyurethane/Eu(MA)3 Films

Europium(III) (Eu³⁺)‐doped polyurethane films were prepared by mixing Eu‐methacrylic acid complex (Eu(MA)₃) with aliphatic polyurethane oligomer and subsequently curing under UV irradiation. Transmission electron microscopy photos and the appearance of the resulting hybrid films showed that phase separation occurred only at an Eu(MA)₃ content above 20 wt.‐%. Fluorescence spectra indicated that the fluorescence of Eu³⁺ was barely influenced by the polyurethane matrix and its intensity increased with an Eu(MA)₃ content in the range of approximately 0 to 10 wt.‐%. An obvious applied external‐field‐dependent magnetization (M) of polyurethane/Eu(MA)₃ films, namely, an increasing M at low field and a decreasing M at high field, was observed at room temperature from the hysteresis loops, which was influenced by both the Eu(MA)₃ content and the ultrasonication imposed on the coatings before curing. It seems that ultrasonication leads to a thermodynamically‐unstable structure of Eu³⁺ in hybrid films, which can be fixed by UV curing but gradually rearranges to its original form during the thermal‐curing process, and enhances the diamagnetic part of the hybrid film. Thus, the magnetic property of Eu³⁺‐doped polyurethane film at room temperature can be adjusted by simply changing the preparation method and the Eu(MA)₃ content instead of the type of Eu³⁺‐organic complex.

     

Frequent alteration of MDM2 and p53 in the molecular progression of recurring non-Hodgkin's lymphoma

AIMS: Recurrence of non-Hodgkin's lymphoma with or without transformation is often associated with increased clinical drug resistance and poor prognosis indicating molecular progression. The study addresses the currently poorly understood molecular mechanisms underlying relapsing non-Hodgkin's lymphoma. METHODS AND RESULTS: We have analysed sequential biopsies from 42 non-Hodgkin's lymphoma patients immunohistochemically for p53 alterations (based on p53 and p21Waf1 expression), as well as for expression of MDM2, p27Kip1 and cyclin D3. Relapse of follicle centre lymphoma was associated with p53 alterations as 5/6 (83%) follicle centre lymphomas with normal p53 at diagnosis showed p53 alterations at relapse. Of these cases, three showed transformation to diffuse large B-cell lymphoma. p53 alteration was also associated with relapse of de novo diffuse large B-cell lymphoma and T-cell non-Hodgkin's lymphoma, as 2/5 (40%) diffuse large B-cell lymphomas and 3/9 (33%) T-cell non-Hodgkin's lymphomas with normal p53 at diagnosis showed p53 alterations at relapse. No indolent non-Hodgkin's lymphoma case showed MDM2 over-expression at diagnosis, whereas 4/5 (80%) transformed diffuse large B-cell lymphomas developed MDM2 over-expression. CONCLUSION: Our data are consistent with the notion that p53 alterations are important for the histological transformation of follicle centre lymphoma. However, the data also suggest that relapsing follicle centre lymphomas without overt transformation often have p53 alterations and increased risk of transformation, and that relapse of de novo diffuse large B-cell lymphomas and T-cell non-Hodgkin's lymphomas is associated with p53 alterations. Furthermore, our results are consistent with an association of MDM2 over-expression with histological transformation of both follicle centre lymphoma and marginal zone B-cell lymphoma.     

Respiratory syncytial virus infection suppresses IFN-gamma production of gammadelta T cells

The immunological mechanisms by which respiratory syncytial virus (RSV) contributes to the development of asthma are poorly understood. gammadelta T cells are important in mucosal defence, and may contribute to the establishment of primary immune responses by producing cytokines early during respiratory infections. Thus, we used flow cytometry and intracellular cytokine staining to investigate the expression of interferon (IFN)-gamma and interleukin (IL)-4 by mitogen-stimulated gammadelta T cells from the peripheral blood of 15 hospitalized infants with RSV bronchiolitis, seven rotavirus-infected infants and eight normal controls. gammadelta T cells from RSV-infected infants had a lower proportion of IFN-gamma-producing cells (median, 4.00%; range, 0.58-6.60%) and a slightly but significantly higher proportion of IL-4-producing cells (median, 0.40%; range, 0.13-2.76%) than rotavirus-infected infants (median, 32.10%; range, 14.43-61.21%; P < 0.01, median, 0.00%; range, 0.00-0.00%; P < 0.05) in the acute phase. By contrast, differences in cytokine production by total CD3+ T cells did not differ significantly between patient groups. Thus, reduced IFN-gamma-production by gammadelta T cells in the peripheral blood of RSV-infected infants is accompanied by increased Th2 cytokine production during the acute phase of disease. At follow-up, eight children had recurrent episodes of wheezing. The frequencies of IFN-gamma-producing gammadelta T cells were significantly lower in patients who developed recurrent wheezing (median, 0.65%; range, 0.02-1.75%) than in patients without recurrent wheezing (median, 6.90%; range, 5.25-10.98%; P < 0.005). Cytokine production by gammadelta T cells may therefore be important in the pathogenesis of acute RSV disease, and play a part in the development of recurrent childhood wheezing after bronchilolitis.     

Functional analysis of α1β1 integrin in human natural killer cells

Upon activation with interleukin (IL)-2 human natural killer (NK) cells acquire on their surface the α1β1 and α2β1 integrins and down-regulate the expression of α6β1. By employing α1β1-specific monoclonal antibody (mAb) HP-2B6, characterized in our laboratory, we examined the functional role of the α1β1 integrin in NK cells. Treatment with HP-2B6 mAb partially interfered with attachment of cultured NK cells to type I collagen, and combined with an anti-α2β1 (TEA 1/41) mAb, it completely abrogated cell adhesion to this extracelular matrix protein. In contrast, NK cell attachment to laminin was completely blocked by the anti-β1 LIA 1/2 mAb, but was unaffected by α1 and α2-specific mAb; as α3β1 and α6β1 were undetectable, the data indicate that the α1β1 integrin binding sites for type I collagen and laminin are different. Incubation with anti-α1 HP-2B6 or its F(ab')₂ fragments specifically induced a rapid homotypic aggregation of NK cells that was dependent on active metabolism, an intact cytoskeleton and the presence of divalent cations (Ca²⁺ and Mg²⁺); homotypic cell adhesion was selectively blocked by anti-CD18, CD11a or CD54 mAb. In addition, stimulation of cultured NK cells with the anti-α1 HP-2B6 enhanced TNF-α production and induced tyrosine phosphorylation of a 110-kDa protein. Pretreatment with specific inhibitors of protein tyrosine kinase (PTK) activity (tyrphostin 25 and herbimycin A) completely abrogated the functional effects induced by the anti-α1 HP-2B6 mAb. Our data show that ligation of the α1β1 integrin positively modulates IL-2-activated NK cell function via a PTK-dependent pathway.     

Early changes of LIFR and gp130 in sciatic nerve and muscle of diabetic mice

Peripheral neuropathy is a common complication of diabetes mediated by alterations of growth factors. Members of the neuropoietic cytokine family, which include IL-6, LIF, and CNTF among others, have been shown to be important regulators of peripheral nerves and the muscles that they innervate. To investigate their potential role in diabetic nerve and muscle, we studied the expression of the shared receptor subunits, LIFR and gp130 in a mouse model of streptozotocin (STZ)-induced diabetes. The results of Western blotting and densitometric analysis showed that both LIFR and gp130 protein expression were increased in diabetic sciatic nerve compared to control mice at early time points following STZ injection. In diabetic gastrocnemius muscle, LIFR and gp130 were increased from 3 days to 24 weeks following STZ injection. In contrast, both LIFR and gp130 protein expression were decreased in diabetic soleus muscle at 3-days post-injection. Our results suggest that hyperglycemia results in changes to nerve and muscle soon after the onset of diabetes and that cytokines may play a role in this process.     

Alpha-actinin-3 deficiency does not significantly alter oxidative enzyme activity in fast human muscle fibres

Aim: In Western European populations, about 18% of all individuals have a complete deficiency of the alpha‐actinin‐3 protein owing to homozygosity for a stop codon mutation (R577X) in the ACTN3 gene. Actn3^?/? knock‐out mice show increased activity of multiple enzymes in the aerobic metabolic pathway in fast muscle fibres. Whether this observation is also present in human XX genotype carriers compared to RR carriers has not been studied in a fibre‐type‐specific approach in humans. The purpose of this study was therefore to compare fibre‐type‐specific oxidative enzyme activity in humans with a different ACTN3 R577X genotype. Methods: Vastus lateralis muscle biopsy samples of 17 XX and 16 RR subjects were used to measure markers of oxidative capacity [cytochrome c oxidase (CYTOX) and succinate dehydrogenase (SDH)] in a fibre‐type‐specific assay using enzyme histochemistry. Results: Cytochrome c oxidase staining showed no significant genotype group differences in type I or type II muscle fibres. Also, we found no significant differences in SDH staining of fast fibres comparing XX and RR carriers. Conclusion: In conclusion, the increase in oxidative enzyme activity of fast muscle fibres, as reported in an Actn3^?/? knock‐out mouse, was not observed in our human samples. Known differences in metabolic characteristics of muscle fibres in rodents compared to humans may in part explain this discrepancy in findings.