Functional role of serotonergic neuromodulation in Aplysia.

The serotonergic metacerebral cell (MCC) of the mollusk Aplysia produces slow synaptic potentials in motor neurons of the buccal muscle, and increases the rate of ongoing rhythmic burst output of the buccal ganglion. In addition, the MCC acts peripherally to enhance the strength of buccal muscle contractions that are produced by firing of motor neurons. The potentiation of contraction is not associated with any detectable changes of resting membrane potential of muscle cells. Although MCC activity produces a small enhancement of excitatory junctional potentials, several experiments clearly indicate that the MCC has a direct potentiating effect on excitation-contraction coupling. The data suggest that potentiation of contraction might be mediated by cAMP. For example, activity of the MCC enchances the rate of accumulation of cAMP in buccal muscle, application of phosphodiesterase resistant analogs of cAMP potentiates muscle contraction, and a phosphodiesterase inhibitor enhances the effect of MCC stimulation. Recordings from free-moving animals indicate that the MCC becomes activated by exposure of the animal to food stimuli, and that the activation parallels the presence of a food-arousal state. Food-arousal is characterized by enhanced strength and increased frequency of biting responses. Both these effects can result from activity of the MCC. Thus, in this system, modulatory synaptic actions function to provide the substrate for a type behavioral modulation.     

Defining the role of laminin-332 in carcinoma

The deadly feature of cancer, metastasis, requires invasion of cells through basement membranes (BM), which normally act as barriers between tissue compartments. In the case of many epithelially-derived cancers (carcinomas), laminin-332 (Ln-332) is a key component of the BM barrier. This review provides a historical examination of Ln-332 from its discovery through identification of its functions in BM and possible role in carcinomas. Current understanding points to distinct roles for the three Ln-332 subunits (α3, β3, γ2) in cell adhesion, extracellular matrix stability, and cell signaling processes in cancer. Given the large number of studies linking Ln-332 γ2 subunit with cancer prognosis, particular attention is given to the crucial role of this subunit in cancer invasion and to the unanswered questions in this area.     

Defining the role of cooperation in early tumor progression

Competition among cells has long been recognized as an important part of carcinogenesis. However, the role of cellular cooperation in cancer has been largely ignored. In this work, we investigated the role of cooperation in early tumor progression using a mathematical and agent-based modeling approach. We hoped to learn how the introduction of cooperative cells into a cell population would affect the dynamics of the system. We modeled the stem cell compartment of tissue using a spatial structure organized into cell patches, with stem cells able to replicate or leave the stem cell compartment through apoptosis or differentiation. The cells could also acquire mutations in three oncogenes and two tumor suppressor genes. Cooperative cells in our model provided a cooperative signal that increased the fitness of their immediate neighbors, but did not affect their own fitness. Running simulations of the model, we found that if cooperative cells are introduced into a cell population, they steadily proliferate and confer a growth advantage to the entire population. This leads us to conclude that providing a cooperative signal is likely to be under positive selective pressure. When cooperative ability and mutation are concurrently present in the same cells, the overall cell population experiences a significant growth advantage, much greater than with cooperation or mutation alone. This growth advantage is diminished if cells with only oncogene/tumor suppressor mutations are also present in the population, suggesting that the optimal scenario for tumor growth would be for cooperative cells to take over a cell population, and then for mutations in oncogenes and tumor suppressors to arise on a background of cooperation. We predict that cooperation is particularly important in the very early stages of carcinogenesis, when tissue is morphologically and histologically normal. Our results have implications for the screening and early diagnosis of cancer.     

Defining the role of essential genes in human disease

A greater understanding of the causes of human disease can come from identifying characteristics that are specific to disease genes. However, a full understanding of the contribution of essential genes to human disease is lacking, due to the premise that these genes tend to cause developmental abnormalities rather than adult disease. We tested the hypothesis that human orthologs of mouse essential genes are associated with a variety of human diseases, rather than only those related to miscarriage and birth defects. We segregated human disease genes according to whether the knockout phenotype of their mouse ortholog was lethal or viable, defining those with orthologs producing lethal knockouts as essential disease genes. We show that the human orthologs of mouse essential genes are associated with a wide spectrum of diseases affecting diverse physiological systems. Notably, human disease genes with essential mouse orthologs are over-represented among disease genes associated with cancer, suggesting links between adult cellular abnormalities and developmental functions. The proteins encoded by essential genes are highly connected in protein-protein interaction networks, which we find correlates with an over-representation of nuclear proteins amongst essential disease genes. Disease genes associated with essential orthologs also are more likely than those with non-essential orthologs to contribute to disease through an autosomal dominant inheritance pattern, suggesting that these diseases may actually result from semi-dominant mutant alleles. Overall, we have described attributes found in disease genes according to the essentiality status of their mouse orthologs. These findings demonstrate that disease genes do occupy highly connected positions in protein-protein interaction networks, and that due to the complexity of disease-associated alleles, essential genes cannot be ignored as candidates for causing diverse human diseases.     

The role of astrocytes in the immunopathogenesis of toxoplasmic encephalitis

Challenge with the parasite Toxoplasma gondii eventually leads to persistent infection characterised by the presence of tissue cysts in the brain of the host. In immunocompetent individuals the parasite rarely leads to disease but in the immunocompromised host reactivation of these cysts can lead to toxoplasmic encephalitis. It is known that both CD4(+) and CD8(+) T cells are important in preventing reactivation of the parasite, however there is also evidence that astrocytes, a subset of glial cells dominant in the CNS, may be important in resistance to T. gondii. The aim of this paper is to review what is known about the immune functions of astrocytes, and the possible role they may play during toxoplasmic encephalitis.     

Defining the role of PfCRT in Plasmodium falciparum chloroquine resistance

Recent studies have highlighted the importance of a parasite protein referred to as the chloroquine resistance transporter (PfCRT) in the molecular basis of Plasmodium falciparum resistance to the quinoline antimalarials. PfCRT, an integral membrane protein with 10 predicted transmembrane domains, is a member of the drug/metabolite transporter superfamily and is located on the membrane of the intra-erythrocytic parasite's digestive vacuole. Specific polymorphisms in PfCRT are tightly correlated with chloroquine resistance. Transfection studies have now proven that pfcrt mutations confer verapamil-reversible chloroquine resistance in vitro and reveal their important role in resistance to quinine. Available evidence is consistent with the view that PfCRT functions as a transporter directly mediating the efflux of chloroquine from the digestive vacuole.     

Defining the role of complement in experimental pemphigus vulgaris in mice

Parenteral passive transfer of human pemphigus vulgaris IgG (PV IgG) into neonatal mice reproduces the cutaneous disease. We used this model to study the role of complement in the development of acantholysis in three steps. Peptic F(ab')2 fragments were prepared from PV IgG and were injected into seven newborn mice, and all animals developed acantholytic skin blisters without local complement activation, as shown by direct immunofluorescence. These fragments were reduced and alkylated to produce Fab' fragments with equivalent in vitro binding activity. The monovalent fragments were given in an identical fashion to five littermates but failed to produce disease even though they were bound in the epidermis in vivo. Intact PV IgG was injected in 20 genetically C5-deficient neonates (B10-D2-OSN strain) and 20 control neonates (B10-D2-NSN, normal complementemic). Extensive blistering, with a positive Nikolsky sign, was produced in all 40 animals. PV IgG was given to 34 BALB/c neonates that were complement depleted by pretreatment with cobra venom factor (CoF) for 24 hr, and to 38 untreated neonates from the same litters. There was no difference in the disease produced after CoF treatment in animals that received high doses of PV IgG (5 to 15 mg/g/day). In animals receiving 2.5 mg PV IgG/g/day, blister formation was delayed and the final extent of the cutaneous lesions was less in CoF-treated mice (n = 12) than in normal complementemic controls (n = 12, p less than 0.02). These results show that complement activation is not an essential mechanism in PV IgG-induced acantholysis in vivo, but it does have an amplifying effect on the development of cutaneous lesions under certain conditions, and lesions can be induced in vivo by bivalent F(ab')2 fragments of PV IgG, but not by the monovalent Fab' fragments, suggesting that cross-linking of the cell surface antigen is an initiating signal in acantholysis.     

The neuropharmacology of emesis: the role of receptors in neuromodulation of nausea and vomiting

The basic pharmacological mechanisms involved in mediating nausea and vomiting are still poorly understood. Several classes of drugs have been identified that alleviate the symptoms of nausea and vomiting, either prophylactically or acutely. None of these is completely effective in all cases. They include antihistamines, dopamine antagonists, steroids, cannabinoids, benzodiazepines, serotonin antagonists, and anticholinergics. This paper examines the evidence that links each of these classes of drugs with the distribution of specific neurotransmitter receptor sites on which they may be acting. Studies on the central nervous system distribution of binding sites for one of these classes of drugs, the anticholinergics, are described. Binding sites for the muscarinic cholinergic radioligand [3H]quinuclidinylbenzilate occur in different concentrations throughout the dorsal vagal complex of the rabbit medulla oblongata. The distribution of such sites in this nonvomiting experimental animal is markedly different from that in the cat, an animal that has been used for many physiological and pharmacological studies of emesis. A previous study has suggested that muscarinic binding sites may occur presynaptically on vagal afferent terminals that synapse in the dorsal vagal complex of the cat; this appears not to be the case in the rabbit. Possible implications of these findings for the identification of the site of action of anticholinergic, antiemetic drugs are discussed.     

Defining the role of the Bcl-2 family proteins in Huntington's disease

B-cell lymphoma 2 (Bcl-2) family proteins regulate survival, mitochondria morphology dynamics and metabolism in many cell types including neurons. Huntington''s disease (HD) is a neurodegenerative disorder caused by an expanded CAG repeat tract in the IT15 gene that encodes for the protein huntingtin (htt). In vitro and in vivo models of HD and HD patients'' tissues show abnormal mitochondrial function and increased cell death rates associated with alterations in Bcl-2 family protein expression and localization. This review aims to draw together the information related to Bcl-2 family protein alterations in HD to decipher their potential role in mutated htt-related cell death and mitochondrial dysfunction.     

Defining the role of arginine 96 in green fluorescent protein fluorophore biosynthesis

Aequoria victoria green fluorescent protein (GFP) is a revolutionary molecular biology tool because of its spontaneous peptide backbone cyclization and chromophore formation from residues Ser65, Tyr66, and Gly67. Here we use structure-based design, comprehensive targeted mutagenesis, and high-resolution crystallography to probe the significant functional role of conserved Arg96 (R96) in chromophore maturation. The R96M GFP variant, in which the R96M side chain is similar in volume but lacks the R96 positive charge, exhibits dramatically slower chromophore maturation kinetics (from hours to months). Comparison of the precyclized conformation of the chromophore-forming residues with the mature R96M chromophore reveals a similar Y66 conformer, contrary to the large Y66 conformational change previously defined in the slowly maturing R96A variant [Barondeau, D. P., Putnam, C. D., Kassmann, C. J., Tainer, J. A., and Getzoff, E. D. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 12111-12116]. Comprehensive R96 mutagenesis and fluorescent colony screening indicate that only the R96K substitution restores wild-type maturation kinetics. Further, we show that the slowly maturing R96A variant can be complemented with a Q183R second-site mutation designed to restore the missing R96 positive charge and rapid fluorophore biosynthesis. Moreover, comparative structural analysis of R96M, R96K, R96A/Q183R, and wild-type GFP reveals the importance of the presence of positive charge, rather than its exact position. Together, these structural, mutational, and biochemical results establish a pivotal role for the R96 positive charge in accelerating the GFP post-translational modification, with implications for peptide backbone cyclization in GFP, its homologues, and related biological systems.     

Defining the role of the Escherichia coli chaperone SecB using comparative proteomics

To improve understanding and identify novel substrates of the cytoplasmic chaperone SecB in Escherichia coli, we analyzed a secB null mutant using comparative proteomics. The secB null mutation did not affect cell growth but caused significant differences at the proteome level. In the absence of SecB, dynamic protein aggregates containing predominantly secretory proteins accumulated in the cytoplasm. Unprocessed secretory proteins were detected in radiolabeled whole cell lysates. Furthermore, the assembly of a large fraction of the outer membrane proteome was slowed down, whereas its steady state composition was hardly affected. In response to aggregation and delayed sorting of secretory proteins, cytoplasmic chaperones DnaK, GroEL/ES, ClpB, IbpA/B, and HslU were up-regulated severalfold, most likely to stabilize secretory proteins during their delayed translocation and/or rescue aggregated secretory proteins. The SecB/A dependence of 12 secretory proteins affected by the secB null mutation (DegP, FhuA, FkpA, OmpT, OmpX, OppA, TolB, TolC, YbgF, YcgK, YgiW, and YncE) was confirmed by "classical" pulse-labeling experiments. Our study more than triples the number of known SecB-dependent secretory proteins and shows that the primary role of SecB is to facilitate the targeting of secretory proteins to the Sec-translocase.     

Defining the role of the MHC in autoimmunity: a review and pooled analysis

The major histocompatibility complex (MHC) is one of the most extensively studied regions in the human genome because of the association of variants at this locus with autoimmune, infectious, and inflammatory diseases. However, identification of causal variants within the MHC for the majority of these diseases has remained difficult due to the great variability and extensive linkage disequilibrium (LD) that exists among alleles throughout this locus, coupled with inadequate study design whereby only a limited subset of about 20 from a total of approximately 250 genes have been studied in small cohorts of predominantly European origin. We have performed a review and pooled analysis of the past 30 years of research on the role of the MHC in six genetically complex disease traits – multiple sclerosis (MS), type 1 diabetes (T1D), systemic lupus erythematosus (SLE), ulcerative colitis (UC), Crohn's disease (CD), and rheumatoid arthritis (RA) – in order to consolidate and evaluate the current literature regarding MHC genetics in these common autoimmune and inflammatory diseases. We corroborate established MHC disease associations and identify predisposing variants that previously have not been appreciated. Furthermore, we find a number of interesting commonalities and differences across diseases that implicate both general and disease-specific pathogenetic mechanisms in autoimmunity.     

Defining the role of ubiquitin-interacting motifs in the polyglutamine disease protein, ataxin-3

Polyglutamine (polyQ) expansions cause neurodegeneration that is associated with protein misfolding and influenced by functional properties of the host protein. The polyQ disease protein, ataxin-3, has predicted ubiquitin-specific protease and ubiquitin-binding domains, which suggest that ataxin-3 functions in ubiquitin-dependent protein surveillance. Here we investigate direct links between the ubiquitin-proteasome pathway and ataxin-3. In neural cells we show that, through its ubiquitin interaction motifs (UIMs), normal or expanded ataxin-3 binds a broad range of ubiquitinated proteins that accumulate when the proteasome is inhibited. The expression of a catalytically inactive ataxin-3 (normal or expanded) causes ubiquitinated proteins to accumulate in cells, even in the absence of proteasome inhibitor. This accumulation of ubiquitinated proteins occurs primarily in the cell nucleus in transfected cells and requires intact UIMs in ataxin-3. We further show that both normal and expanded ataxin-3 can undergo oligoubiquitination. Although this post-translational modification occurs in a UIM-dependent manner, it becomes independent of UIMs when the catalytic cysteine residue of ataxin-3 is mutated, suggesting that ataxin-3 ubiquitination is itself regulated in trans by its own de-ubiquitinating activity. Finally, pulse-chase labeling reveals that ataxin-3 is degraded by the proteasome, with expanded ataxin-3 being as efficiently degraded as normal ataxin-3. Mutating the UIMs does not alter degradation, suggesting that UIM-mediated oligoubiquitination of ataxin-3 modulates ataxin-3 function rather than stability. The function of ataxin-3 as a de-ubiquitinating enzyme, its post-translational modification by ubiquitin, and its degradation via the proteasome link this polyQ protein to ubiquitin-dependent pathways already implicated in disease pathogenesis.     

Ethanol-induced oxidative stress in rat astrocytes: role of HSP70

Ethanol intake is associated with increase in lipid peroxidation and formation of reactive oxygen species in different cerebral areas, in neurons as well as in astrocytes. The latter's integrity is essential for the normal growth of neurons. In previous studies we observed, in different cerebral areas of both acutely and chronically ethanol-treated rats, correlation between ethanol-induced oxidative stress and the increased expression of HSP70 (70 kDa heat shock proteins), chaperonins having a protective and stabilizing effect on stress–induced cell injury. In this study we examined, in vitro, the role of HSP70 on chronically ethanol-treated rat astrocytes by transfection with an anti-HSP70 antisense oligonucleotide. The results show that treatment with ethanol, from 50 to 100 mmol/L, induces a dose-dependent increase in the production of reactive oxygen species and of HSP70 levels, together with an impairment of the respiratory chain activity and a decrease in cell viability. In addition, our data indicate a drastic reduction of cellular metabolism in HSP70-deprived astrocytes, particularly when these cells were also ethanol-treated. In fact, transfection with HSP70 antisense induced moderate oxidative damage in control astrocytes and, consequently, a drastic decrease in the viability of ethanol-treated cells, with the mitochondrial functionality being particularly affected. Our results confirm that heat shock proteins confer a survival advantage to the astrocytes, preventing oxidative damage and nuclear DNA damage as well, and suggest the development of new drugs exerting a cytoprotective role either in physiological, or pathological conditions. This revised version was published online in July 2006 with corrections to the Cover Date.     

Defining a new role of GW182 in maintaining miRNA stability

GW182 binds to Argonaute (AGO) proteins and has a central role in miRNA‐mediated gene silencing. Using lentiviral shRNA‐induced GW182 knockdown in HEK293 cells, this study identifies a new role of GW182 in regulating miRNA stability. Stably knocking down GW182 or its paralogue TNRC6B reduces transfected miRNA‐mimic half‐lives. Replenishment of GW182 family proteins, as well as one of its domain Δ12, significantly restores the stability of transfected miRNA‐mimic. GW182 knockdown reduces miRNA secretion via secretory exosomes. Targeted siRNA screening identifies a 3′–5′ exoribonuclease complex responsible for the miRNA degradation only when GW182 is knocked down. Immunoprecipitation further confirms that the presence of GW182 in the RISC complex is critical in protecting Argonaute‐bound miRNA.