Search

The Wnt signaling pathway induces various responses underlying the development and maturation of the nervous system. Wnt ligands are highly hydrophobic proteins that limit their diffusion through an aqueous extracellular medium to a target cell. Nevertheless, their attachment to small extracellular vesicles-like exosomes is one of the described mechanisms that allow their transport under this condition. Some Wnt ligands in these vehicles are expected to be dependent on post-translational modifications such as acylation. The mechanisms determining Wnt loading in exosomes and delivery to the target cells are largely unknown. Here, we took advantage of a cell model that secret a highly enriched population of small extracellular vesicles (sEVs), hippocampal HT-22 neurons. First, to establish the cell model, we characterized the morphological and biochemical properties of an enriched fraction of sEVs obtained from hippocampal HT-22 neurons that express NCAM-L1, a specific exosomal neuronal marker. Transmission electron microscopy showed a highly enriched fraction of exosome-like vesicles. Next, the exosomal presence of Wnt3a, Wnt5a, and Wnt7a was confirmed by western blot analysis and electron microscopy combined with immunogold. Also, we studied whether palmitoylation is a necessary post-translational modification for the transport Wnt in these vesicles. We found that proteinase-K treatment of exosomes selectively decreased their Wnt5a and Wnt7a content, suggesting that their expression is delimited to the exterior membrane surface. In contrast, Wnt3a remained attached, suggesting that it is localized within the exosome lumen. On the other hand, Wnt-C59, a specific inhibitor of porcupine O-acyltransferase (PORCN), decreased the association of Wnt with exosomes, suggesting that Wnt ligand acylation is necessary for them to be secreted by exosomes. These findings may help to understand the action of the Wnt ligands in the target cell, which could be defined during the packaging of the ligands in the secretory cell sEVs.

Dysregulated Wnt signaling is linked to major neurodegenerative diseases, including Alzheimer disease (AD). In mouse models of AD, activation of the canonical Wnt signaling pathway improves learning/memory, but the mechanism for this remains unclear. The decline in brain function in AD patients correlates with reduced glucose utilization by neurons. Here, we test whether improvements in glucose metabolism mediate the neuroprotective effects of Wnt in AD mouse model. APPswe/PS1dE9 transgenic mice were used to model AD, Andrographolide or Lithium was used to activate Wnt signaling, and cytochalasin B was used to block glucose uptake. Cognitive function was assessed by novel object recognition and memory flexibility tests. Glucose uptake and the glycolytic rate were determined using radiotracer glucose. The activities of key enzymes of glycolysis such as hexokinase (HK) and phosphofructokinase (PFK), Adenosine triphosphate (ATP)/ Adenosine diphosphate (ADP) levels and the pentose phosphate pathway (PPP) and activity of glucose-6 phosphate dehydrogenase were measured. Wnt activators significantly improved brain glucose utilization and cognitive performance in transgenic mice. Wnt signaling enhanced glucose metabolism by increasing the expression and/or activity of HK, PFK and AMP-activated protein kinase (AMPK). Inhibiting glucose uptake partially abolished the beneficial effects of Wnt signaling on learning/memory. Wnt activation also enhanced glucose metabolism in cortical and hippocampal neurons, as well as brain slices derived from APPswe/PS1E9 transgenic mice. Combined, these data provide evidence that the neuroprotective effects of Wnt signaling in AD mouse models result, at least in part, from Wnt-mediated improvements in neuronal glucose metabolism.

Among all the biological systems in vertebrates, the central nervous system (CNS) is the most complex, and its function depends on specialized contacts among neurons called synapses. The assembly and organization of synapses must be exquisitely regulated for a normal brain function and network activity. There has been a tremendous effort in recent decades to understand the molecular and cellular mechanisms participating in the formation of new synapses and their organization, maintenance, and regulation. At the vertebrate presynapses, proteins such as Piccolo, Bassoon, RIM, RIM-BPs, CAST/ELKS, liprin-α, and Munc13 are constant residents and participate in multiple and dynamic interactions with other regulatory proteins, which define network activity and normal brain function. Here, we review the function of these active zone (AZ) proteins and diverse factors involved in AZ assembly and maintenance, with an emphasis on axonal trafficking of precursor vesicles, protein homo- and hetero-oligomeric interactions as a mechanism of AZ trapping and stabilization, and the role of F-actin in presynaptic assembly and its modulation by Wnt signaling.

Alzheimer's disease (AD) is the most common type of dementia. The onset and progression of this pathology are correlated with several changes in the brain, including the formation of extracellular aggregates of amyloid-beta (Aβ) peptide and the intracellular accumulation of hyperphosphorylated tau protein. In addition, dysregulated neuronal plasticity, synapse loss, and a reduction in cellular energy metabolism have also been described. Canonical Wnt signaling has also been shown to be downregulated in AD. Remarkably, we showed previously that the in vivo inhibition of Wnt signaling accelerates the appearance of AD markers in transgenic (Tg) and wild-type (WT) mice. Additionally, we found that Wnt signaling stimulates energy metabolism, which is critical for the ability of Wnt to promote the recovery of cognitive function in AD. Therefore, we hypothesized that activation of canonical Wnt signaling in a presymptomatic transgenic animal model of AD would improve some symptoms. To explore the latter, we used a transgenic mouse model (J20 Tg) with mild AD phenotype expression (high levels of amyloid aggregates) and studied the effect of andrographolide (ANDRO), an activator of canonical Wnt signaling. We found that presymptomatic administration of ANDRO in J20 Tg mice prevented the reduction in cellular energy metabolism markers. Moreover, treated animals showed improvement in cognitive performance. At the synaptic level, J20 Tg animals showed severe deficiencies in presynaptic function as determined by electrophysiological parameters, all of which were completely restored to normal by ANDRO administration. Finally, an analysis of hippocampal synaptosomes by electron microscopy revealed that the length of synapses was restored with ANDRO treatment. Altogether, these data support the idea that the activation of canonical Wnt signaling during presymptomatic stages could represent an interesting pharmacological strategy to delay the onset of AD.

Purpose To assess the effects of terpenic denture cleanser on denture biofilm removal using scanning electron microscopy (SEM). Materials and Methods The internal surface biofilm of four maxillary dentures was elucidated with Caristop-revelador Dual Tone, and 40 blue-stained specimens (0.6 cm × 0.4 cm × 2 mm) were obtained. These specimens were randomly assigned to one of the following four groups of 10 specimens each: control, Eci Clean, Fitty Dent, and terpenic denture cleanser. The period of immersion in each solution was 12 hours. Biofilm removal was evaluated using SEM, and morphologically varying areas of the SEM images were quantified with Imaris software. The data were analyzed using Kolmogorov-Smirnov, t-tests, ANOVA, and Tamhane's tests (p = 0.05). Results Data revealed that terpenic denture cleanser removed significantly more biofilm than any other treatment examined in this study. The t-tests revealed significant differences in the clean area that resulted from the use of the terpenic cleanser compared with the clean area that resulted from the use of Eci Clean (p = 0.013). Fitty Dent was the least effective and left dirty acrylic resin. The average areas with few removed layers were 59.3%, 43.3%, and 9.5% in Fitty Dent, Eci Clean, and terpenic cleanser groups, respectively. Tamhane's tests indicated that the Eci Clean and Fitty Dent groups were significantly different from the 0.5% terpenic cleanser group (p = 0.008). Conclusion The terpenic denture cleanser was effective in removing denture biofilm.

Wnt ligands play critical roles in neuronal development, synapse formation, synaptic activity, and plasticity. Synaptic plasticity requires molecular remodeling of synapses, implying the expression of key synaptic components. Some studies have linked Wnt signaling activity to changes in synaptic protein levels. However, the presynaptic and postsynaptic gene expression profiles of hippocampal neurons exposed to Wnt proteins have not been studied. Hence, we treated rat cultured hippocampal neurons with recombinant Wnt3a, lithium, and the Wnt inhibitor Dkk-1 for different treatment durations and measured the mRNA and protein levels of pre- and postsynaptic components. The ligand Wnt3a promoted the differential temporal expression of genes encoding presynaptic and postsynaptic proteins. Gene expression of the presynaptic proteins Rim1, piccolo (Pclo), Erc2, Ctbp1 and Rimbp2 increased in a specific temporal pattern. Simultaneously, the mRNA and protein levels of postsynaptic components showed a different temporal expression pattern, e.g., the mRNAs for postsynaptic scaffolding components such as postsynaptic density protein-95 (PSD-95/Dlg4), Homer1 and Shank1 were temporally regulated by both Wnt3a and lithium. On the other hand, the mRNA levels of the gene encoding the protein calcium/calmodulin-dependent protein kinase IV (Camk4), canonically upregulated by Wnt, were increased. Our results suggest that Wnt signaling orchestrates expressional changes in genes encoding presynaptic and postsynaptic components, probably as part of a synaptic plasticity mechanism in neurons.

Communication between cells occurs through secreted molecules, among which Wnt ligands play a critical role in balancing cell proliferation, differentiation and cellular homeostasis. The action of Wnt signaling can be modulated at several levels, including posttranslational modification of the Wnt ligands, whose acylation is critical for biological activity. At least three enzymes are necessary for Wnt acylation/deacylation: stearoyl CoA desaturase (SCD), porcupine (PORCN) and Notum. At the endoplasmic reticulum (ER), SCD provides the monounsaturated fatty acid to PORCN, which adds it to the Wnt ligand; at the extracellular matrix, the fatty acid is removed by Notum. Obviously, the interplay between these enzymes will define Wnt signaling ligand secretion and activity. Excessive activation of Wnt signaling has been observed in a variety of solid tumors, which has led the pharmaceutical industry to develop specific inhibitors for this pathway that mainly target PORCN, some of which are in early clinical trials. In the central nervous system (CNS), Wnt signaling activation has been shown to have a neuroprotective effect, and conversely, its inhibition induces neurodegeneration, which implies that the inhibition of PORCN in cancer therapies should be used with caution, and the cognitive performance of the patient should be monitored during treatment. This review collects information about the PORCN enzyme in relation to its role in the Wnt pathway through the acylation of Wnt ligands, its inhibition by drugs in the treatment of some cancers, and its putative modulation in the treatment of neurodegenerative diseases.

Neurons are highly polarized cells displaying an elaborate architectural morphology. The design of their dendritic arborization and the distribution of their synapses contribute importantly to information processing in the brain. The growth and complexity of dendritic arbors are driven by the formation of synapses along their lengths. Synaptogenesis is augmented by the secretion of factors, like BDNF, Reelin, BMPs, or Wnts. Exo70 is a component of the exocyst complex, a protein complex that guides membrane addition and polarized exocytosis. While it has been linked to cytokinesis and the establishment of cell polarity, its role in synaptogenesis is poorly understood. In this report, we show that Exo70 plays a role in the arborization of dendrites and the development of synaptic connections between cultured hippocampal neurons. Specifically, while the overexpression of Exo70 increases dendritic arborization, synapse number, and spine density, the inhibition of Exo70 expression reduces secondary and tertiary dendrite formation and lowers synapse density. Moreover, increasing Exo70 expression augmented synaptic vesicle recycling as evaluated by FM4-64 dye uptake and the inverse was observed with downregulation of endogenous Exo70. Monitoring the formation of dendritic spines by super-resolution microscopy, we also observed that mRFP-Exo70 accumulates at the tip of EGFP-β-actin-positive filopodia. Together, these results suggest that Exo70 is essentially involved in the formation of synapses and neuronal dendritic morphology.

Synapses are complex structures that allow communication between neurons in the central nervous system. Studies conducted in vertebrate and invertebrate models have contributed to the knowledge of the function of synaptic proteins. The functional synapse requires numerous protein complexes with specialized functions that are regulated in space and time to allow synaptic plasticity. However, their interplay during neuronal development, learning, and memory is poorly understood. Accumulating evidence links synapse proteins to neurodevelopmental, neuropsychiatric, and neurodegenerative diseases. In this review, we describe the way in which several proteins that participate in cell adhesion, scaffolding, exocytosis, and neurotransmitter reception from presynaptic and postsynaptic compartments, mainly from excitatory synapses, have been associated with several synaptopathies, and we relate their functions to the disease phenotype.

HIV/AIDS continues to expand throughout the world, and in recent times has had a severe impact on the number of women living with HIV. Access to methods of prevention for HIV/AIDS around the world is limited and for many women may not be feasible. Examples of such methods are sexual abstinence, which though recognized as an effective method of prevention is difficult for many women to achieve. Mutual fidelity is another option, but many women cannot control the sexual behavior of their partners. Finally, the male condom continues to be an available method of HIV prevention for sexually active people. However, in many places, gender inequality, social norms, and economic disparities severely limit women's capacity to negotiate the use of a condom with their partner. For this reason, an urgent need exists to develop a product for the prevention of HIV/AIDS that can be handled by women. Microbicides, products that can reduce HIV risk when applied intravaginally, hold promise for stopping the advance of HIV/AIDS, especially when considering that women can make their own decisions about whether to use them when other methods of prevention are not available. The objective of the current literature review was to understand the scientific advances related to microbicides in the prevention of HIV/AIDS in women through the analysis of available literature in this area. Interest in this topic emerged from the need to contribute to women's health and HIV/AIDS prevention. To achieve this review's objective, a search was carried out in multiple databases, including OVID, PUBMED, PROQUEST, and CINAHL, as well as published materials from organizations related to this area such as UNAIDS and CONASIDA, and literature available from the Internet. Upon completion of the literature review, it was concluded that microbicides are an effective method for preventing HIV/AIDS for women as well as their partners. Although they have a high level of acceptance among the female population, the majority of microbicides are still in clinical studies, requiring further evaluation for their safe use in humans. There are 3 microbicides in the final phases of clinical studies that will soon be available on the market. One of these was found to be 60% effective in protecting against the transmission of HIV and was used by women in 50% of their sexual activity, which resulted in avoiding approximately 2.5 million new HIV infections in men, women, and children over a 3-year period.El VIH/SIDA sigue expandiéndose a nivel mundial y en el último tiempo ha tenido un impacto severo en el número de mujeres que viven con VIH. El acceso a métodos de prevención para VIH/SIDA alrededor del mundo son limitados y no factibles de acceder para muchas mujeres. Dentro de estos métodos se encuentran: la abstinencia sexual que es reconocida como un efectivo método de prevención para VIH, pero es considerada por las mujeres como difícil de realizar, en segundo lugar esta la fidelidad mutua en la relación de pareja, donde muchas mujeres no pueden asegurar el comportamiento sexual de sus parejas, y por ultimo el preservativo masculino que continua siendo el método de elección disponible para la prevención de VIH/SIDA en personas sexualmente activas, sin embargo, en muchos lugares, las desigualdades de género, normas sociales y disparidades económicas, limitan severamente la capacidad de las mujeres a negociar el uso del preservativo con sus parejas. Es por esta razón, que existe la urgente necesidad de desarrollar un producto para la prevención de VIH/SIDA que sea manejado por mujeres. Los Microbicidas, productos que pueden reducir el riesgo de VIH, cuando son aplicados en forma intravaginal, representan una promesa para frenar el avance del VIH/SIDA, sobretodo considerando que las mujeres podrán tomar sus propias desiciones para utilizarlos o no cuando todas las otras medidas de prevención no sean posibles de utilizar. Es por este motivo, que la presente revisión bibliográfica tiene como propósito conocer el avance científico sobre el desarrollo de los microbicidas en la prevención de VIH/SIDA en mujeres, a través del análisis de la literatura disponible respecto al tema. El interés en este tema surge a partir de la necesidad de contribuir a la salud de las mujeres y a la prevención del VIH/SIDA. Para responder este propósito se realizó una búsqueda de información a través de distintas bases de datos, entre ellas OVID, PUBMED, PROQUEST y CINAHL, además de material publicado en organizaciones relacionadas con el tema ONUSIDA, CONASIDA y literatura accesible en Internet. Al finalizar esta revisión bibliográfica, se concluye que los microbicidas se presentan como un método efectivo para prevenir el VIH/SIDA, tanto en mujeres como en sus parejas. Presentan una alta aceptabilidad en la población femenina, sin embargo la mayoría de los candidatos a microbicidas aún se encuentran en estudios clínicos, necesitando una mayor evaluación para que su uso en humanos sea seguro. Tres son los microbicidas que están prontamente a finalizar las fases de estudio y estar disponibles en el mercado. Un microbicida que fuese 60% efectivo para proteger contra el VIH y fuese utilizado por las mujeres en un 50% de su actividad sexual, se evitarían en 3 años 2.5 millones de infecciones por VIH (en mujeres, hombres y niños).

Synaptic vesicles (SVs) fuse with the plasma membrane at a precise location called the presynaptic active zone (AZ). This fusion is coordinated by proteins embedded within a cytoskeletal matrix assembled at the AZ (CAZ). In the present study, we have identified a novel binding partner for the CAZ proteins Piccolo and Bassoon. This interacting protein, Trio, is a member of the Dbl family of guanine nucleotide exchange factors (GEFs) known to regulate the dynamic assembly of actin and growth factor dependent axon guidance and synaptic growth. Trio was found to interact with the C-terminal PBH 9/10 domains of Piccolo and Bassoon via its own N-terminal Spectrin repeats, a domain that is also critical for its localization to the CAZ. Moreover, our data suggest that regions within the C-terminus of Trio negatively regulate its interactions with Piccolo/Bassoon. These findings provide a mechanism for the presynaptic targeting of Trio and support a model in which Piccolo and Bassoon play a role in regulating neurotransmission through interactions with proteins, including Trio, that modulate the dynamic assembly of F-actin during cycles of synaptic vesicle exo- and endocytosis.

Neurotransmitter release from presynaptic nerve terminals is restricted to specialized areas of the plasma membrane, so-called active zones. Active zones are characterized by a network of cytoplasmic scaffolding proteins involved in active zone generation and synaptic transmission. To analyze the modes of biogenesis of this cytomatrix, we asked how Bassoon and Piccolo, two prototypic active zone cytomatrix molecules, are delivered to nascent synapses. Although these proteins may be transported via vesicles, little is known about the importance of a vesicular pathway and about molecular determinants of cytomatrix molecule trafficking. We found that Bassoon and Piccolo co-localize with markers of the trans-Golgi network in cultured neurons. Impairing vesicle exit from the Golgi complex, either using brefeldin A, recombinant proteins, or a low temperature block, prevented transport of Bassoon out of the soma. Deleting a newly identified Golgi-binding region of Bassoon impaired subcellular targeting of recombinant Bassoon. Overexpressing this region to specifically block Golgi binding of the endogenous protein reduced the concentration of Bassoon at synapses. These results suggest that, during the period of bulk synaptogenesis, a primordial cytomatrix assembles in a trans-Golgi compartment. They further indicate that transport via Golgi-derived vesicles is essential for delivery of cytomatrix proteins to the synapse. Paradigmatically this establishes Golgi transit as an obligatory step for subcellular trafficking of distinct cytoplasmic scaffolding proteins.

Recent studies indicate that active zones (AZs)—sites of neurotransmitter release—may be assembled from preassembled AZ precursor vesicles inserted into the presynaptic plasma membrane. Here we report that one putative AZ precursor vesicle of CNS synapses—the Piccolo-Bassoon transport vesicle (PTV)—carries a comprehensive set of AZ proteins genetically and functionally coupled to synaptic vesicle exocytosis. Time-lapse imaging reveals that PTVs are highly mobile, consistent with a role in intracellular transport. Quantitative analysis reveals that the Bassoon, Piccolo, and RIM content of individual PTVs is, on average, half of that of individual presynaptic boutons and shows that the synaptic content of these molecules can be quantitatively accounted for by incorporation of integer numbers (typically two to three) of PTVs into presynaptic membranes. These findings suggest that AZs are assembled from unitary amounts of AZ material carried on PTVs.

Mutagenesis of recombinant rho1 gamma-aminobutyric acid (GABA) receptors has previously identified five residues in the amino terminal extracellular domain that play an important role in GABA binding. Here, we present evidence that the tyrosine at position 102 of the rho1 receptor is also associated with the agonist binding site. Wild-type and mutant rho1 receptors were expressed in Xenopus laevis oocytes and examined using the two-electrode voltage clamp. When Tyr-102 was mutated to cysteine, serine, tryptophan, or glycine the EC(50) increased 31-, 214-, 664-, and 8752-fold, respectively. An increase in the IC(50) was also observed for the competitive antagonist 3-APMPA, but not for the non-competitive antagonist picrotoxin. Y102C was accessible to modification by methanethiosulfonate, and this modification was prevented by both GABA and 3-APMPA. An interesting characteristic of the Y102S mutant receptor was that, in the absence of GABA, there was an unusually high oocyte resting conductance that was blocked by both 3-APMPA and picrotoxin, indicating spontaneously opening GABA receptors. It appears that mutation of Tyr-102 perturbs the binding site and gates the pore. We conclude that Tyr-102 is a component of the GABA binding domain and speculate that Tyr-102 might be important for coupling agonist binding to channel opening.

Vesicular trafficking of presynaptic and postsynaptic components is emerging as a general cellular mechanism for the delivery of scaffold proteins, ion channels, and receptors to nascent and mature synapses. However, the molecular mechanisms leading to the selection of cargos and their differential transport to subneuronal compartments are not well understood, in part because of the mixing of cargos at the plasma membrane and/or within endosomal compartments. In the present study, we have explored the cellular mechanisms of active zone precursor vesicle assembly at the Golgi in dissociated hippocampal neurons ofRattus norvegicus. Our studies show that Piccolo, Bassoon, and ELKS2/CAST exit thetrans-Golgi network on a common vesicle that requires Piccolo and Bassoon for its proper assembly. In contrast, Munc13 and synaptic vesicle proteins use distinct sets of Golgi-derived transport vesicles, while RIM1α associates with vesicular membranes in a post-Golgi compartment. Furthermore, Piccolo and Bassoon are necessary for ELKS2/CAST to leave the Golgi in association with vesicles, and a core domain of Bassoon is sufficient to facilitate formation of these vesicles. While these findings support emerging principles regarding active zone differentiation, the cellular and molecular analyses reported here also indicate that the Piccolo-Bassoon transport vesicles leaving the Golgi may undergo further changes in protein composition before arriving at synaptic sites.

Arsenic main inorganic compound is arsenic trioxide (ATO) presented in solution mainly as arsenite. ATO increases intracellular pH (pHi), cell proliferation and tumor growth. Sodium-proton exchangers (NHEs) modulate the pHi, with NHE1 playing significant roles. Whether ATO-increased cell proliferation results from altered NHEs expression and activity is unknown. We hypothesize that ATO increases cell proliferation by altering pHi due to increased NHEs-like transport activity. Madin-Darby canine kidney (MDCK) cells grown in 5 mmol/L D-glucose-containing DMEM were exposed to ATO (0.05, 0.5 or 5 µmol/L, 0-48 hours) in the absence or presence of 5-N,N-hexamethylene amiloride (HMA, 5-100 µmol/L, NHEs inhibitor), PD-98059 (30 µmol/L, MAPK1/2 inhibitor), Gö6976 (10 µmol/L, PKCα, βI and μ inhibitor), or Schering 28080 (10 µmol/L, H(+)/K(+)ATPase inhibitor) plus concanamycin (0.1 µmol/L, V type ATPases inhibitor). Incorporation of [(3)H]thymidine was used to estimate cell proliferation, and counting cells with a hemocytometer to determine the cell number. The pHi was measured by fluorometry in 2,7-bicarboxyethyl-5,6-carboxyfluorescein loaded cells. The Na(+)-dependent HMA-sensitive NHEs-like mediated proton transport kinetics, NHE1 protein abundance in the total, cytoplasm and plasma membrane protein fractions, and phosphorylated and total p42/44 mitogen-activated protein kinases (p42/44(mapk)) were also determined. Lowest ATO (0.05 µmol/L, ~0.01 ppm) used in this study increased cell proliferation, pHi, NHEs-like transport and plasma membrane NHE1 protein abundance, effects blocked by HMA, PD-98059 or Gö6976. Cell-buffering capacity did not change by ATO. The results show that a low ATO concentration increases MDCK cells proliferation by NHEs (probably NHE1)-like transport dependent-increased pHi requiring p42/44(mapk) and PKCα, βI and/or μ activity. This finding could be crucial in diseases where uncontrolled cell growth occurs, such as tumor growth, and in circumstances where ATO, likely arsenite, is available at the drinking-water at these levels.

Presynaptic differentiation takes place over three interrelated acts involving the biogenesis and trafficking of molecular complexes of active zone material, the "trapping" or stabilization of active zone sites, and the subsequent development of mature synapses. Although the identities of proteins involved with establishing presynaptic specializations have been increasingly delineated, the exact functional mechanisms by which the active zone is assembled remain poorly understood. Here, we discuss a theoretical model for how the trapping stage of presynaptic differentiation might occur in developing neurons. We suggest that subsets of active zone proteins containing polyglutamine domains undergo concentration-dependent prion-like conversions as they accumulate at the plasma membrane. This conversion might serve to aggregate the proteins into a singular structure, which is then able to recruit scaffolding agents necessary for regulated synaptic transmission. A brief informatics analysis in support of this 'Q' assembly hypothesis--across commonly used models of synaptogenesis--is presented.

Classic IRES sequences are notorious for exerting biased expression in favor of upstream coding regions when placed into polycistronic vectors. Here, we report the development of a bicistronic lentiviral system based on the 1D/2A sequence from the foot-and-mouth disease virus that is able to maintain tightly balanced control of upstream and downstream protein expression for several days at a stoichiometry very closely approaching 1.0. Our results suggest that the 1D/2A sequence can be optimized in an FUGW lentiviral setting to coordinate expression of multiple polypeptides, presenting a potentially valuable tool to signaling network researchers and to the gene therapy community.

1. 1. Very few mitochondria, undetectable levels of succinate dehydrogenase and glutamate dehydrogenase and significant activities of glutamate pyruvate transaminase, glutamate oxaloacetate transaminase and malate dehydrogenase were observed in the deeper foot muscle tissue of Concholepas concholepas. 2. 2. Malate dehydrogenase activity was resolved into two molecular forms, which differ in Km values for oxaloacetate and malate, and sensitivity to inhibition by α-ketoglutarate. 3. 3. In addition to reoxidation of NADH generated from glycolysis, the function of malate dehydrogenase is discussed in connection with octopine synthesis and oxidation in the foot muscle of C. concholepas. 4. 4. The results of initial rate and product inhibition studies of both forms of malate dehydrogenase are consistent with an Ordered Bi Bi kinetic mechanism, with NADH adding first and NAD+ leaving last. Oxaloacetate substrate inhibition is suggested to result from formation of an enzyme-NAD+-oxaloacetate dead end complex.