Neuronal calcium signaling: function and dysfunction

Calcium (Ca²⁺) is an universal second messenger that regulates the most important activities of all eukaryotic cells. It is of critical importance to neurons as it participates in the transmission of the depolarizing signal and contributes to synaptic activity. Neurons have thus developed extensive and intricate Ca²⁺ signaling pathways to couple the Ca²⁺ signal to their biochemical machinery. Ca²⁺ influx into neurons occurs through plasma membrane receptors and voltage-dependent ion channels. The release of Ca²⁺ from the intracellular stores, such as the endoplasmic reticulum, by intracellular channels also contributes to the elevation of cytosolic Ca²⁺. Inside the cell, Ca²⁺ is controlled by the buffering action of cytosolic Ca²⁺-binding proteins and by its uptake and release by mitochondria. The uptake of Ca²⁺ in the mitochondrial matrix stimulates the citric acid cycle, thus enhancing ATP production and the removal of Ca²⁺ from the cytosol by the ATP-driven pumps in the endoplasmic reticulum and the plasma membrane. A Na⁺/Ca²⁺ exchanger in the plasma membrane also participates in the control of neuronal Ca²⁺. The impaired ability of neurons to maintain an adequate energy level may impact Ca²⁺ signaling: this occurs during aging and in neurodegenerative disease processes. The focus of this review is on neuronal Ca²⁺ signaling and its involvement in synaptic signaling processes, neuronal energy metabolism, and neurotransmission. The contribution of altered Ca²⁺ signaling in the most important neurological disorders will then be considered.     

Allosteric function and dysfunction of the prion protein

Transmissible spongiform encephalopathies (TSEs) are neurodegenerative diseases associated with progressive oligo- and multimerization of the prion protein (PrP^C), its conformational conversion, aggregation and precipitation. We recently proposed that PrP^C serves as a cell surface scaffold protein for a variety of signaling modules, the effects of which translate into wide-range functional consequences. Here we review evidence for allosteric functions of PrP^C, which constitute a common property of scaffold proteins. The available data suggest that allosteric effects among PrP^C and its partners are involved in the assembly of multi-component signaling modules at the cell surface, impose upon both physiological and pathological conformational responses of PrP^C, and that allosteric dysfunction of PrP^C has the potential to entail progressive signal corruption. These properties may be germane both to physiological roles of PrP^C, as well as to the pathogenesis of the TSEs and other degenerative/non-communicable diseases.     

Congenital disorders involving defective N-glycosylation of proteins

This review deals with several of the main autosomal recessive congenital disorders involving defective N-glycosylation of proteins (the addition of glycans linked to the polypeptide chain by a beta-linkage between the anomeric carbon of N-acetylglucosamine and the amido group of L-asparagine). These congenital disorders of glycosylation (CDG, previously known as carbohydrate-deficient glycoprotein syndromes) are a group of multisystemic diseases often involving severe psychomotor retardation. Six distinct variants of CDG in group I (types Ia-If) have been described to date and the defects have been localized to deficiencies in the assembly of the dolichylpyrophosphate-linked oligosaccharide N-glycan precursor and its transfer to asparagine residues on the nascent polypeptides. Two variants of CDG group II (types IIa and IIb) have been identified as defects in the processing of protein-bound N-glycans. Hereditary erythroblastic multinuclearity with a positive acidified-serum lysis test (HEMPAS; congenital dyserythropoietic anemia type II) presents as a relatively mild dyserythropoietic anemia. The genetic defect in most cases of HEMPAS is not known, but alpha-3/6-mannosidase II is involved in at least some patients. Leukocyte adhesion deficiency type II (LAD II) is a rare disorder characterized by recurrent infections, persistent leukocytosis and severe mental and growth retardation. LAD II is due to lack of availability of GDP-fucose. The study of these diseases and of relevant animal models has provided strong evidence that N-glycans are essential for normal mammalian development.     

Regulation of antibody effector functions through IgG Fc N-glycosylation

Immunoglobulin gamma (IgG) antibodies are key effector proteins of the immune system. They recognize antigens with high specificity and are indispensable for immunological memory following pathogen exposure or vaccination. The constant, crystallizable fragment (Fc) of IgG molecules mediates antibody effector functions such as complement-dependent cytotoxicity, antibody-mediated cellular cytotoxicity, and antibody-dependent cell-mediated phagocytosis. These functions are regulated by a single N-linked, biantennary glycan of the heavy chain, which resides just below the hinge region, and the presence of specific sugar moieties on the glycan has profound implications on IgG effector functions. Emerging knowledge of how Fc glycans contribute to IgG structure and functions has opened new avenues for the therapeutic exploitation of defined antibody glycoforms in the treatment of cancer and autoimmune diseases. Here, we review recent advances in understanding proinflammatory IgG effector functions and their regulation by Fc glycans.     

Modulation of connexin signaling by bacterial pathogens and their toxins

Inherent to their pivotal tasks in the maintenance of cellular homeostasis, gap junctions, connexin hemichannels, and pannexin hemichannels are frequently involved in the dysregulation of this critical balance. The present paper specifically focuses on their roles in bacterial infection and disease. In particular, the reported biological outcome of clinically important bacteria including Escherichia coli, Shigella flexneri, Yersinia enterocolitica, Helicobacter pylori, Bordetella pertussis, Aggregatibacter actinomycetemcomitans, Pseudomonas aeruginosa, Citrobacter rodentium, Clostridium species, Streptococcus pneumoniae, and Staphylococcus aureus and their toxic products on connexin- and pannexin-related signaling in host cells is reviewed. Particular attention is paid to the underlying molecular mechanisms of these effects as well as to the actual biological relevance of these findings.     

Modulation of antibody synthesis by an anti-tumour alga

Summary An unicellular alga,Chlorella pyrenoidosa, which had been reported to protect C₃H mice against sarcoma BP8, is shown, when injected in Freund's incomplete adjuvant, to modulate the antibody synthesis induced by immunization with a hapten-carrier complex.C. pyrenoidosa appeared to be able to initiate an antigenic competition between hapten and carrier determinants of the antigen molecule during antibody synthesis, and thus it could be speculated thatC. pyrenoidosa modulates the immune response at the macrophage level.Acknowledgments. This work was supported by the Secrétariat d'Etat aux Universités and by the DGRST, grant No. 77.71347.     

Modulation of SMN nuclear foci and cytoplasmic localization by its C-terminus

The survival of motor neuron ( SMN1) gene product, SMN, is detected both in the cytoplasm and in nuclear gems and cajal bodies. We show here that SMN exon 6 is essential both for formation of its nuclear foci and for its cytoplasmic localization. However, exon 7 inhibits the formation of SMN nuclear foci but promotes SMN cytoplasmic localization. More interestingly, we find that a random C-terminal tag of five or more amino acids downstream of exon 6 is sufficient to inhibit the occurrence of multiple nuclear foci and to promote cytoplasmic localization of SMNDelta7, the primary product of the SMN2 gene. Moreover, SMNDelta7 proteins that bear spinal muscular atrophy mutations in exon 6 either showed defects in nuclear foci formation or enhanced cytoplasmic localization. We conclude that exon 6 and exon 7 synergistically regulate SMN distribution that may require specific exon 6 motifs but is independent of specific sequences in exon 7.     

Modulation by flavonoids of cell multidrug resistance mediated by P-glycoprotein and related ABC transporters

Cancer cell resistance to chemotherapy is often mediated by overexpression of P-glycoprotein, a plasma membrane ABC (ATP-binding cassette) transporter which extrudes cytotoxic drugs at the expense of ATP hydrolysis. P-glycoprotein (ABCB1, according to the human gene nomenclature committee) consists of two homologous halves each containing a transmembrane domain (TMD) involved in drug binding and efflux, and a cytosolic nucleotide-binding domain (NBD) involved in ATP binding and hydrolysis, with an overall (TMD-NBD)2 domain topology. Homologous ABC multidrug transporters, from the same ABCB family, are found in many species such as Plasmodiumfalciparum and Leishmania spp. protozoa, where they induce resistance to antiparasitic drugs. In yeasts, some ABC transporters involved in resistance to fungicides, such as Saccharomyces cerevisiae Pdr5p and Snq2p, display a different (NBD-TMD)2 domain topology and are classified in another family, ABCG. Much effort has been spent to modulate multidrug resistance in the different species by using specific inhibitors, but generally with little success due to additional cellular targets and/or extrusion of the potential inhibitors. This review shows that due to similarities in function and maybe in three-dimensional organization of the different transporters, common potential modulators have been found. An in vitro 'rational screening' was performed among the large flavonoid family using a four-step procedure: (i) direct binding to purified recombinant cytosolic NBD and/or full-length transporter, (ii) inhibition of ATP hydrolysis and energy-dependent drug interaction with transporter-enriched membranes, (iii) inhibition of cell transporter activity monitored by flow cytometry and (iv) chemosensitization of cell growth. The results indicate that prenylated flavonoids bind with high affinity, and strongly inhibit drug interaction and nucleotide hydrolysis. As such, they constitute promising potential modulators of multidrug resistance.     

Modulation of transmission in different electronic junctions by aminopyridine

Summary 4 distinct electronic inputs can be identified in amphibian motoneurons perfused with calcium-free solution containing manganese. The differential effect of 4-aminopyridine on different electrotonic junctions may reflect the peculiarities of molecular architecture of potassium channels in different electrically excitable presynaptic membranes.We thank Prof. D. R. Curtis, of National University Canberra, for the gift of 4-aminopyridine.     

E-cadherin和MMP-2在大肠癌中的表达及其意义

目的 探讨大肠癌组织中E-钙粘素(E-cadherin)和基质金属蛋白酶-2(MMP-2)的表达及意义.方法 应用免疫组织化学技术检测92例大肠癌及20例正常大肠组织中E-cadherin和MMP-2的表达,同时结合患者的临床病理资料进行分析.结果 E-cadherin在正常大肠组织中的阳性表达率为85％ (17/20),高于大肠癌组织中52.2％ (48/92)的表达率,两者具有显著差异性(p＜0.01).MMP-2在大肠癌组织中的表达率为71.7％ (70/92),显著高于正常肠组织中35％ (7/20)的表达率(p＜0.01).E-cadherin 和MMP-2的表达都与大肠癌患者性别、年龄无明显相关,但与肿瘤浸润深度、临床分期、淋巴结转移密切相关(P＜0.05);两者的表达具有负相关(r=-0.316,P ＜0.05).结论 E-cadherin和MMP-2与大肠癌的发生发展密切相关.E-cadhein的正常表达将显著降低大肠癌的浸润和转移能力.而MMP-2蛋白阳性表达则促进大肠癌浸润和转移.E-cadherin和MMP-2的检测可以成为临床判断大肠癌的生物学行为的重要参考指标.     

Modulation of transmission in different electronic junctions by aminopyridine.

4 distinct electronic inputs can be identified in amphibian motoneurons perfused with calcium-free solution containing manganese. The differential effect of 4-aminopyridine on different electronic junctions may reflect the peculiarities of molecular architecture of potassium channels in different electrically excitable presynaptic membranes.     

Modulation of phospholipase A2 activity generated by molecular evolution

Snake venom oligomeric neurotoxins offer several unique examples of modulation of phospholipase A2 (PLA2) activity generated by molecular evolution. This phenomenon was found in evolutionary younger snakes and is probably common for representatives of the genus Vipera. At present, the best-studied example is the heterodimeric neurotoxin vipoxin from the venom of the southeast European snake Vipera ammodytes meridionalis. It is a complex between a basic strongly toxic PLA2 and an acidic and catalytically inactive PLA2-like component (Inh). This is the first reported example of a high degree of structural homology (62%) between an enzyme and its natural protein inhibitor. The inhibitor is a product of the divergent evolution of the unstable PLA2 in order to stabilize it and to preserve the pharmacological activity/toxicity for a long time. Inh reduces both the catalytic activity and toxicity of PLA2. Vipoxin also illustrates evolution of the catalytic into a inhibitory function. Vipoxin analogues have been found in the venom of viperid snakes inhabiting diverse regions of the world. An attempt is made to explain modulation of the toxic function by the three-dimensional structure of vipoxin.     

Human mesenchymal stem cells induce E-cadherin degradation in breast carcinoma spheroids by activating ADAM10

Mesenchymal stem cells (MSCs) have been shown to communicate with tumor cells. We analyzed the effect of human MSCs (hMSCs) on breast cancer cells in three-dimensional cultures. By using GFP expression and immunohistochemistry, we show that hMSCs invade 3D breast cancer cell aggregates. hMSCs caused breast cancer spheroids to become disorganized which was accompanied by a disruption of cell–cell adhesion, E-cadherin cleavage, and nuclear translocation of E-cadherin, but not by epithelial/mesenchymal transition or by an increase in ERK1/2 activity. In addition, hMSCs enhanced the motility of breast cancer cells. Inhibition of ADAM10 (a disintegrin and metalloprotease 10), known to cleave E-cadherin, prevented both hMSC-mediated E-cadherin cleavage and enhanced migration. Our data suggest that hMSCs interfere with cell–cell adhesion and enhance migration of breast cancer cells by activating ADAM10.     

Modulation of natural killer cell cytotoxicity and cytokine release by the drug glatiramer acetate

Glatiramer acetate (GA or Copaxone) is a drug used to treat experimental autoimmune encephalomyelitis in mice and multiple sclerosis in human. Here, we describe a new mechanism of action for this drug. GA enhanced the cytolysis of human NK cells against autologous and allogeneic immature and mature monocyte-derived dendritic cells (DCs). This drug reduced the percentages of mature DCs expressing CD80, CD83, HLA-DR or HLA-I. In contrast, it did not modulate the percentages of NK cells expressing NKG2D, NKp30, or NKp44. Nonetheless, anti-NKp30 or anti-CD86 inhibited GA-enhanced human NK cell lysis of immature DCs. Hence, CD86, and NKp30 are important for NK cell lysis of immature DCs, whereas CD80, CD83, HLA-DR and HLA-I are important for the lysis of mature DCs when GA is used as a stimulus. Further, GA inhibited the release of IFN-γ 24 h but increased the release of TNF-α 48 h after incubation with NK cells. Received 13 November 2008; received after revision 10 February 2009; accepted 18 February 2009     

Modulation of protein biophysical properties by chemical glycosylation: biochemical insights and biomedical implications

Glycosylation constitutes one of the most important posttranslational modifications employed by biological systems to modulate protein biophysical properties. Due to the direct biochemical and biomedical implications of achieving control over protein stability and function by chemical means, there has been great interest in recent years towards the development of chemical strategies for protein glycosylation. Since current knowledge about glycoprotein biophysics has been mainly derived from the study of naturally glycosylated proteins, chemical glycosylation provides novel insights into its mechanistic understanding by affording control over glycosylation parameters. This review presents a survey of the effects that natural and chemical glycosylation have on the fundamental biophysical properties of proteins (structure, dynamics, stability, and function). This is complemented by a mechanistic discussion of how glycans achieve such effects and discussion of the implications of employing chemical glycosylation as a tool to exert control over protein biophysical properties within biochemical and biomedical applications. Received 15 December 2006; received after revision 28 March 2007; accepted 25 April 2007     

Cerebrovascular reactivity to CO2: Modulation by arterial pressure

Summary Cerebrovascular reactivity to CO₂ (CO₂R), measured in halothane-anesthetized rabbits, decreased as arterial pressure was increased either pharmacologically or mechanically. On the other hand, hypotension, induced by bleeding, led to an increase in CO₂R. These responses were unaffected by denervation of baroreceptors.This work was supported by grants from NIH (HL 17903) and American Heart Association — Greater Los Angeles Affiliate (437IG). To whom requests for reprints should be sent.     

Modulation by polyamines of apoptotic pathways triggered by procyanidins in human metastatic SW620 cells

We showed previously that inhibition of polyamine catabolism with the polyamine oxidase inhibitor MDL 72527 (MDL) potentiates the apoptotic effects of apple procyanidins (Pcy) in SW620 cells. Here we report that Pcy caused an activation of the intrinsic apoptotic pathway through enhanced polyamine catabolism and mitochondrial membrane depolarization. MDL in the presence of Pcy caused a profound intracellular depletion of polyamines and exerted a protective effect on mitochondrial functions. MDL potentiation of Pcy-triggered apoptosis was reversed by addition of exogenous polyamines. In addition, MDL in combination with Pcy activated the extrinsic apoptotic pathway through enhanced TRAIL-death receptor (DR4/DR5) expression. Potentiation of Pcy-triggered apoptosis by MDL was inhibited when cells were exposed to specific inhibitors of DR4/DR5. These data indicate that the depletion of intracellular polyamines by MDL in the presence of Pcy caused a switch from intrinsic to extrinsic apoptotic pathways in human colon cancer-derived metastatic cells. Received 15 January 2008; received after revision 19 February 2008; accepted 7 March 2008