Regulation of progranulin expression in human microglia and proteolysis of progranulin by matrix metalloproteinase-12 (MMP-12)

Background

The essential role of progranulin (PGRN) as a neurotrophic factor has been demonstrated by the discovery that haploinsufficiency due to GRN gene mutations causes frontotemporal lobar dementia. In addition to neurons, microglia in vivo express PGRN, but little is known about the regulation of PGRN expression by microglia.

Goal

In the current study, we examined the regulation of expression and function of PGRN, its proteolytic enzyme macrophage elastase (MMP-12), as well as the inhibitor of PGRN proteolysis, secretory leukocyte protease inhibitor (SLPI), in human CNS cells.

Methods

Cultures of primary human microglia and astrocytes were stimulated with the TLR ligands (LPS or poly IC), Th1 cytokines (IL-1/IFNγ), or Th2 cytokines (IL-4, IL-13). Results were analyzed by Q-PCR, immunoblotting or ELISA. The roles of MMP-12 and SLPI in PGRN cleavage were also examined.

Results

Unstimulated microglia produced nanogram levels of PGRN, and PGRN release from microglia was suppressed by the TLR ligands or IL-1/IFNγ, but increased by IL-4 or IL-13. Unexpectedly, while astrocytes stimulated with proinflammatory factors released large amounts of SLPI, none were detected in microglial cultures. We also identified MMP-12 as a PGRN proteolytic enzyme, and SLPI as an inhibitor of MMP-12-induced PGRN proteolysis. Experiments employing PGRN siRNA demonstrated that microglial PGRN was involved in the cytokine and chemokine production following TLR3/4 activation, with its effect on TNFα being the most conspicuous.

Conclusions

Our study is the first detailed examination of PGRN in human microglia. Our results establish microglia as a significant source of PGRN, and MMP-12 and SLPI as modulators of PGRN proteolysis. Negative and positive regulation of microglial PGRN release by the proinflammatory/Th1 and the Th2 stimuli, respectively, suggests a fundamentally different aspect of PGRN regulation compared to other known microglial activation products. Microglial PGRN appears to function as an endogenous modulator of innate immune responses.     

Novel Insights into the Echinoderm Nervous System from Histaminergic and FMRFaminergic-Like Cells in the Sea Cucumber Leptosynapta clarki

Understanding of the echinoderm nervous system is limited due to its distinct organization in comparison to other animal phyla and by the difficulty in accessing it. The transparent and accessible, apodid sea cucumber Leptosynapta clarki provides novel opportunities for detailed characterization of echinoderm neural systems. The present study used immunohistochemistry against FMRFamide and histamine to describe the neural organization in juvenile and adult sea cucumbers. Histaminergic- and FMRFaminergic-like immunoreactivity is reported in several distinct cell types throughout the body of L. clarki. FMRFamide-like immunoreactive cell bodies were found in the buccal tentacles, esophageal region and in proximity to the radial nerve cords. Sensory-like cells in the tentacles send processes toward the circumoral nerve ring, while unipolar and bipolar cells close to the radial nerve cords display extensive processes in close association with muscle and other cells of the body wall. Histamine-like immunoreactivity was identified in neuronal somatas located in the buccal tentacles, circumoral nerve ring and in papillae distributed across the body. The tentacular cells send processes into the nerve ring, while the processes of cells in the body wall papillae extend to the surface epithelium and radial nerve cords. Pharmacological application of histamine produced a strong coordinated, peristaltic response of the body wall suggesting the role of histamine in the feeding behavior. Our immunohistochemical data provide evidence for extensive connections between the hyponeural and ectoneural nervous system in the sea cucumber, challenging previously held views on a clear functional separation of the sub-components of the nervous system. Furthermore, our data indicate a potential function of histamine in coordinated, peristaltic movements; consistent with feeding patterns in this species. This study on L. clarki illustrates how using a broader range of neurotransmitter systems can provide better insight into the anatomy, function and evolution of echinoderm nervous sytems.     

HIV-1 Expressing the Envelopes of Transmitted/Founder or Control/Reference Viruses Have Similar Infection Patterns of CD4 T-Cells in Human Cervical Tissue Ex Vivo

Recently, it was found that 80% of sexual HIV-1 transmissions are established by a single virion/viral genome. To investigate whether the transmitted/founder (T/F) viruses have specific biological properties favoring sexual transmission, we inoculated human cervical tissue explants with isogenic HIV-1 viruses encoding Env sequences from T/F and control reference (C/R) HIV-1 variants as well as with full length T/F HIV-1 and compared their replication efficiencies, T cell depletion, and the activation status of infected cells. We found that all the HIV-1 variants were capable of transmitting infection to cervical tissue ex vivo and in this system preferentially replicate in activated CD4 T cells and deplete these cells. There was no difference in the biological properties of T/F and C/R HIV-1 variants as evaluated in ex vivo cervical tissue.     

Characterization of novel diesel-degrading strains <Emphasis Type="Italic">Acinetobacter haemolyticus</Emphasis> MJ01 and <Emphasis Type="Italic">Acinetobacter johnsonii</Emphasis> MJ4 isolated from oil-contaminated soil

The diesel-degrading strains, designated as MJ01 and MJ4, were isolated from oil-contaminated soil in Daejeon (South Korea) and were taxonomically characterized using a polyphasic approach and their diesel oil degradation abilities were analyzed. The isolates MJ01 and MJ4 were identified as Acinetobacter haemolyticus and Acinetobacter johnsonii, respectively, based on their 16S rDNA gene sequences, DNA–DNA relatedness, fatty acid profiles and various physiological characteristics. Strains MJ01 and MJ4 were able to use diesel oil as the sole carbon and energy source. Both strains could degrade over 90% of diesel oil with an initial concentration of 20,000 mg/l after incubation for 7 days, the most significant degradation occurred during the first 3 days. To our knowledge, this is the first report on diesel oil-degrading microorganisms among bacterial strains belonging to A. haemolyticus and A. johnsonii.     

Biochemical characterization of WbdN, a β1,3-glucosyltransferase involved in O-antigen synthesis in enterohemorrhagic Escherichia coli O157

The enterohemorrhagic O157 strain of Escherichia coli, which is one of the most well-known bacterial pathogens, has an O-antigen repeating unit structure with the sequence [-2-d-Rha4NAcα1-3-l-Fucα1-4-d-Glcβ1-3-d-GalNAcα1-]. The O-antigen gene cluster of E. coli O157 contains the genes responsible for the assembly of this repeating unit and includes wbdN. In spite of cloning many O-antigen genes, biochemical characterization has been done on very few enzymes involved in O-antigen synthesis. In this work, we expressed the wbdN gene in E. coli BL21, and the His-tagged protein was purified. WbdN activity was characterized using the donor substrate UDP-[(14)C]Glc and the synthetic acceptor substrate GalNAcα-O-PO(3)-PO(3)-(CH(2))(11)-O-Ph. The enzyme product was isolated by high pressure liquid chromatography, and mass spectrometry showed that one Glc residue was transferred to the acceptor by WbdN. Nuclear magnetic resonance analysis of the product structure indicated that Glc was β1-3 linked to GalNAc. WbdN contains a conserved DxD motif and requires divalent metal ions for full activity. WbdN activity has an optimal pH between 7 and 8 and is highly specific for UDP-Glc as the donor substrate. GalNAcα derivatives lacking the diphosphate group were inactive as substrates, and the enzyme did not transfer Glc to GlcNAcα-O-PO(3)-PO(3)-(CH(2))(11)-O-Ph. Our results illustrate that WbdN is a specific UDP-Glc:GalNAcα-diphosphate-lipid β1,3-Glc-transferase. The enzyme is a target for the development of inhibitors to block O157-antigen synthesis.     

Double rotation of the opening (closing) elytra in beetles (Coleoptera)

Transient movements of the elytra (opening and closing) were filmed in beetles tethered from below. A total of 39 specimens of 18 species representing 11 families were examined. Bright markers glued to the elytra were traced frame by frame. Body-fixed 3D traces of apical and shoulder markers were reconstructed. Shapes of traces reflected different steps of elytral movement and different types of flight. Flat circular arcs were fitted to scattered traces using the least square method. The rotation axis of the apical marker was always directed at the contralateral side. The trace of the shoulder marker was, as a rule, non-parallel to the apical trace. Usually, the shoulder marker on the costal edge of the elytron uniformly supinated in the course of adduction of the apical marker. Traces of opening and closing coincided, hence the double rotation of the elytron had one degree of freedom. The elytron to body articulation in beetles is, presumably, a spherical mechanism with two separate but linked drives for a broad swing during opening (closing).     

Sequence,Structure, and Evolution of Cellulases in Glycoside Hydrolase Family 48

Currently, the cost of cellulase enzymes remains a key economic impediment to commercialization of biofuels (1). Enzymes from glycoside hydrolase family 48 (GH48) are a critical component of numerous natural lignocellulose-degrading systems. Although computational mining of large genomic data sets is a promising new approach for identifying novel cellulolytic activities, current computational methods are unable to distinguish between cellulases and enzymes with different substrate specificities that belong to the same protein family. We show that by using a robust computational approach supported by experimental studies, cellulases and non-cellulases can be effectively identified within a given protein family. Phylogenetic analysis of GH48 showed non-monophyletic distribution, an indication of horizontal gene transfer. Enzymatic function of GH48 proteins coded by horizontally transferred genes was verified experimentally, which confirmed that these proteins are cellulases. Computational and structural studies of GH48 enzymes identified structural elements that define cellulases and can be used to computationally distinguish them from non-cellulases. We propose that the structural element that can be used for in silico discrimination between cellulases and non-cellulases belonging to GH48 is an ω-loop located on the surface of the molecule and characterized by highly conserved rare amino acids. These markers were used to screen metagenomics data for “true” cellulases.     

Effects of acetylcholinesterase inhibitor paraoxon denote the possibility of non-quantal acetylcholine release in myocardium of different vertebrates

Effects of organophosphorous acetylcholinesterase inhibitor paraoxon were studied in the isolated atrial and ventricular myocardium preparations of a fish (cod), an amphibian (frog) and a mammal (rat) using the microelectrode technique. Incubation of isolated atrium with paraoxon (5 × 10⁻⁶–5 × 10⁻⁵ M) caused significant reduction of action potential duration and marked slowing of sinus rhythm. These effects were abolished by muscarinic blocker atropine and therefore are caused by acetylcholine, which accumulates in the myocardium due to acetylcholinesterase inhibition even in the absence of vagal input. Hemicholinium III is a blocker of high affinity choline-uptake transporters, which are believed to mediate non-quantal release of acetylcholine from cholinergic terminals in different tissues. In the atrial myocardium of all the three studied species, hemicholinium III (10⁻⁵ M) significantly suppressed all the effects of paraoxon. Blocker of parasympathetic ganglionic transmission hexamethonium bromide (10⁻⁴ M) and inhibitor of vesicular acetylcholine transporters vesamicol (10⁻⁵ M) failed to attenuate paraoxon effects. Among ventricular myocardium preparations of three species paraoxon provoked marked cholinergic effects only in frog, hemicholinium III abolished these effects effectively. We conclude that paraoxon stops degradation of acetylcholine in the myocardium and helps to reveal the effects of acetylcholine, which is continuously secreted from the cholinergic nerves in non-quantal manner. Thus, non-quantal release of acetylcholine in the heart is not specific only for mammals, but is also present in the hearts of different vertebrates.     

Microbicides: still a long road to success

The development of efficient microbicides, the topically applied compounds that protect uninfected individuals from acquiring HIV-1, is a promising strategy to contain HIV-1 epidemics. Such microbicides should of course possess anti-HIV-1 activity, but they should also act against other genital pathogens, which facilitate HIV-1 transmission. The new trend in microbicide strategy is to use drugs currently used in HIV-1 therapy. The success of this strategy is mixed so far and is impaired by our limited knowledge of the basic mechanisms of HIV-1 transmission as well as by the inadequacy of the systems in which microbicides are tested in preclinical studies.     

Plant phenolics regulate neoplastic cell growth and survival: a quantitative structure-activity and biochemical analysis

The anti-tumour activities of many plant phenolics at high concentrations (>100 micromol/L) suggest their potential use as dietary supplements in cancer chemoprevention and cancer chemotherapy. However, it is not clear what impact phenolic compounds have at the physiological concentrations obtained through consumption of high phenolic diets on neoplastic cells. In the present study, 54 naturally occurring phenolics were evaluated at physiologically relevant concentrations for their capacity to alter PC12 cell viability in response to serum deprivation, the chemotherepeutic agent etoposide, and the apoptogen C2-ceramide. Surprisingly, novel mitogenic, cytoprotective, and antiapoptotic activities were detected. Quantitative structure-activity relationship modelling indicated that many of these activities could be predicted by compound lipophilicity, steric bulk, and (or) antioxidant capacity, with the exception of inhibition of ceramide-induced apoptosis. Where quantitative structure-activity relationship analysis was insufficient, biochemical assessment demonstrated that the benzoate orsellinic acid blocked downstream caspase-12 activation following ceramide challenge. These findings demonstrate substantive mitogenic, cytoprotective, and antiapoptotic biological activities of plant phenolics on neoplastic cells at physiologically relevant dietary concentrations that should be considered in chemopreventive and chemotherapeutic strategies.