The PDZ-GEF Gef26 regulates synapse development and function via FasII and Rap1 at the Drosophila neuromuscular junction

Guanine nucleotide exchange factors (GEFs) are essential for small G proteins to activate their downstream signaling pathways, which are involved in morphogenesis, cell adhesion, and migration. Mutants of Gef26, a PDZ-GEF (PDZ domain-containing guanine nucleotide exchange factor) in Drosophila, exhibit strong defects in wings, eyes, and the reproductive and nervous systems. However, the precise roles of Gef26 in development remain unclear. In the present study, we analyzed the role of Gef26 in synaptic development and function. We identified significant decreases in bouton number and branch length at larval neuromuscular junctions (NMJs) in Gef26 mutants, and these defects were fully rescued by restoring Gef26 expression, indicating that Gef26 plays an important role in NMJ morphogenesis. In addition to the observed defects in NMJ morphology, electrophysiological analyses revealed functional defects at NMJs, and locomotor deficiency appeared in Gef26 mutant larvae. Furthermore, Gef26 regulated NMJ morphogenesis by regulating the level of synaptic Fasciclin II (FasII), a well-studied cell adhesion molecule that functions in NMJ development and remodeling. Finally, our data demonstrate that Gef26-specific small G protein Rap1 worked downstream of Gef26 to regulate the level of FasII at NMJs, possibly through a βPS integrin-mediated signaling pathway. Taken together, our findings define a novel role of Gef26 in regulating NMJ development and function.     

Genomic organization and evolution of the soybean SB92 satellite sequence

Repetitive DNA sequences comprise a large percentage of plant genomes, and their characterization provides information about both species and genome evolution. We have isolated a recombinant clone containing a highly repeated DNA element (SB92) that is homologous to ca. 0.9% of the soybean genome or about 10⁵ copies. This repeated sequence is tandemly arranged and is found in four or five major genomic locations. FISH analysis of metaphase chromosomes suggests that two of these locations are centromeric. We have determined the sequence of two cloned repeats and performed genomic sequencing to obtain a consensus sequence. The consensus repeat size was 92 bp and exhibited an average of 10% nucleotide substitution relative to the two cloned repeats. This high level of sequence diversity suggests an ancient origin but is inconsistent with the limited phylogenetic distribution of SB92, which is found an high copy number only in the annual soybeans. It therefore seems likely that this sequence is undergoing very rapid evolution.     

A two-dimensional proteome map of maize endosperm

We have established a proteome reference map for maize (Zea mays L.) endosperm by means of two-dimensional gel electrophoresis and protein identification with LC-MS/MS analysis. This investigation focussed on proteins in major spots in a 4-7 pI range and 10-100 kDa M(r) range. Among the 632 protein spots processed, 496 were identified by matching against the NCBInr and ZMtuc-tus databases (using the SEQUEST software). Forty-two per cent of the proteins were identified against maize sequences, 23% against rice sequences and 21% against Arabidopsis sequences. Identified proteins were not only cytoplasmic but also nuclear, mitochondrial or amyloplastic. Metabolic processes, protein destination, protein synthesis, cell rescue, defense, cell death and ageing are the most abundant functional categories, comprising almost half of the 632 proteins analyzed in our study. This proteome map constitutes a powerful tool for physiological studies and is the first step for investigating the maize endosperm development.     

Hepatitis C virus core protein enhances HIV-1 replication in human macrophages through TLR2, JNK,and MEK1/2-dependent upregulation of TNF-α and IL-6

Despite their differential cell tropisms, HIV-1 and HCV dramatically influence disease progression in coinfected patients. Macrophages are important target cells of HIV-1. We hypothesized that secreted HCV core protein might modulate HIV-1 replication. We demonstrate that HCV core significantly enhances HIV-1 replication in human macrophages by upregulating TNF-α and IL-6 via TLR2-, JNK-, and MEK1/2-dependent pathways. Furthermore, we show that TNF-α and IL-6 secreted from HCV core-treated macrophages reactivates monocytic U1 cells latently infected with HIV-1. Our studies reveal a previously unrecognized role of HCV core by enhancing HIV-1 infection in macrophages.     

Recent advances in plasmid-based tools for establishing novel microbial chassis

A key challenge for domesticating alternative cultivable microorganisms with biotechnological potential lies in the development of innovative technologies. Within this framework, a myriad of genetic tools has flourished, allowing the design and manipulation of complex synthetic circuits and genomes to become the general rule in many laboratories rather than the exception. More recently, with the development of novel technologies such as DNA automated synthesis/sequencing and powerful computational tools, molecular biology has entered the synthetic biology era. In the beginning, most of these technologies were established in traditional microbial models (known as chassis in the synthetic biology framework) such as Escherichia coli and Saccharomyces cerevisiae, enabling fast advances in the field and the validation of fundamental proofs of concept. However, it soon became clear that these organisms, although extremely useful for prototyping many genetic tools, were not ideal for a wide range of biotechnological tasks due to intrinsic limitations in their molecular/physiological properties. Over the last decade, researchers have been facing the great challenge of shifting from these model systems to non-conventional chassis with endogenous capacities for dealing with specific tasks. The key to address these issues includes the generation of narrow and broad host plasmid-based molecular tools and the development of novel methods for engineering genomes through homologous recombination systems, CRISPR/Cas9 and other alternative methods. Here, we address the most recent advances in plasmid-based tools for the construction of novel cell factories, including a guide for helping with “build-your-own” microbial host.     

Light Activation of Rhodopsin: Insights from Molecular Dynamics Simulations Guided by Solid-State NMR Distance Restraints

Structural restraints provided by solid-state NMR measurements of the metarhodopsin II intermediate are combined with molecular dynamics simulations to help visualize structural changes in the light activation of rhodopsin. Since the timescale for the formation of the metarhodopsin II intermediate (> 1 ms) is beyond that readily accessible by molecular dynamics, we use NMR distance restraints derived from ¹³C dipolar recoupling measurements to guide the simulations. The simulations yield a working model for how photoisomerization of the 11-cis retinylidene chromophore bound within the interior of rhodopsin is coupled to transmembrane helix motion and receptor activation. The mechanism of activation that emerges is that multiple switches on the extracellular (or intradiscal) side of rhodopsin trigger structural changes that converge to disrupt the ionic lock between helices H3 and H6 on the intracellular side of the receptor.     

Effects of molybdate,sulfide, and tetrathiomolybdate on copper metabolism in rats

Species differences in the response to dietary MoO₄^2? as a metabolic antagonist of Cu are considered briefly. Suggestions that (i) the potency of MoO₄^2? as a Cu antagonist is enhanced by normally innocuous dietary concentrations of S^2? and (ii) that MoS₄^2? may be a more effective antagonist than either MoO₄^2? or S^2? were investigated in a series of studies with rats. Diets including MoS₄^2? but not of MoO₄^2? or S^2? alone promoted a decline in hepatic Cu and ceruloplasmin activity and induced clinical signs of Cu deficiency. Evidence of concurrent anomalies in the partition of Cu between tissues and in the distribution of Cu between proteins of plasma and kidney cytosol suggested that such effects were partly attributable to the development of systemic defects in Cu metabolism. The relationship of such findings to the suggested involvement of MoS₄^2? or its derivatives in the etiology of Mo-induced Cu deficiency in ruminant animals is considered.     

Isolation and characterization of fluorescence-enhancing RNA tags

Methods for the visualization of RNAs are urgently needed for studying a wide variety of cellular processes. Here we report on-bead screening of RNA libraries and its application to the isolation of specific fluorescence-enhancing RNA sequences. A one-bead-one-compound combinatorial RNA library with over one million different sequences was synthesized using the split-and-mix method. Solid-phase synthesis of 30 mer RNAs was performed on 15 ??m and 60 ??m diameter polystyrene beads bearing a non-cleavable linker. The RNA-derivatized beads were incubated with the well-established FlAsH pre-fluorophore and then screened for fluorescence enhancement, either by manually picking the brightest beads under a fluorescence microscope or by sorting with a FACS instrument. A protocol was established for sequence determination from single beads. While numerous RNA sequences showed increased fluorescence when immobilized, only few of them influenced the fluorescence properties of the FlAsH dye when detached from the beads. One of these sequences was found to induce a bathochromic shift in the excitation (from 492 to 510 nm) and emission (from 512 to 523 nm) maxima. This shift was accompanied by a 3.6-fold fluorescence enhancement of FlAsH fluorescence intensity. Mutation studies on the sequence revealed a rather robust structural motif.