TIE-2/VEGF-R2 SAR and in vitro activity of C3-acyl dihydroindazolo[5,4-a]pyrrolo[3,4-c]carbazole analogs

Orally bioavailable, dual inhibitors of TIE-2/VEGF-R2 were identified by elaborating the C3/N13 SAR around a fused pyrrolodihydroindazolocarbazole scaffold. Analogs bearing a C3-thiophencarbonyl group were evaluated in enzymatic and cellular biochemical assays; two orally bioavailable analogs were further profiled in functional assays and found to inhibit microvessel growth in rat aortic explant cultures and inhibit Ang-1-stimulated chemotaxis of HUVECs.     

Znf179 E3 ligase-mediated TDP-43 polyubiquitination is involved in TDP-43- ubiquitinated inclusions (UBI) (+)-related neurodegenerative pathology

Background

The brain predominantly expressed RING finger protein, Znf179, is known to be important for embryonic neuronal differentiation during brain development. Downregulation of Znf179 has been observed in motor neurons of adult mouse models for amyotrophic lateral sclerosis (ALS), yet the molecular function of Znf179 in neurodegeneration has never been previously described. Znf179 contains the classical C3HC4 RING finger domain, and numerous proteins containing C3HC4 RING finger domain act as E3 ubiquitin ligases. Hence, we are interested to identify whether Znf179 possesses E3 ligase activity and its role in ALS neuropathy.

Methods

We used in vivo and in vitro ubiquitination assay to examine the E3 ligase autoubiquitination activity of Znf179 and its effect on 26S proteasome activity. To search for the candidate substrates of Znf179, we immunoprecipitated Znf179 and subjected to mass spectrometry (MS) analysis to identify its interacting proteins. We found that ALS/ FTLD-U (frontotemporal lobar degeneration (FTLD) with ubiquitin inclusions)-related neurodegenerative TDP-43 protein is the E3 ligase substrate of Znf179. To further clarify the role of E3 ubiquitin ligase Znf179 in neurodegenerative TDP-43-UBI (ubiquitinated inclusions) (+) proteinopathy, the effect of Znf179-mediated TDP-43 polyubiquitination on TDP-43 protein stability, aggregate formation and nucleus/cytoplasm mislocalization were evaluated in vitro cell culture system and in vivo animal model.

Results

Here we report that Znf179 is a RING E3 ubiquitin ligase which possesses autoubiquitination feature and regulates 26S proteasome activity through modulating the protein expression levels of 19S/20S proteasome subunits. Our immunoprecipitation assay and MS analysis results revealed that the neuropathological TDP-43 protein is one of its E3 ligase substrate. Znf179 interactes with TDP-43 protein and mediates polyubiquitination of TDP-43 in vitro and in vivo. In neurodegenerative TDP-43 proteinopathy, we found that Znf179-mediated polyubiquitination of TDP-43 accelerates its protein turnover rate and attenuates insoluble pathologic TDP-43 aggregates, while knockout of Znf179 in mouse brain results in accumulation of insoluble TDP-43 and cytosolic TDP-43 inclusions in cortex, hippocampus and midbrain regions.

Conclusions

Here we unveil the important role for the novel E3 ligase Znf179 in TDP-43-mediated neuropathy, and provide a potential therapeutic strategy for combating ALS/ FTLD-U neurodegenerative pathologies.

     

Lipid production by microalgae <Emphasis Type="Italic">Chlorella protothecoides</Emphasis> with volatile fatty acids (VFAs) as carbon sources in heterotrophic cultivation and its economic assessment

Volatile fatty acids (VFAs) that can be derived from food wastes were used for microbial lipid production by Chlorella protothecoides in heterotrophic cultures. The usage of VFAs as carbon sources for lipid accumulation was investigated in batch cultures. Culture medium, culture temperature, and nitrogen sources were explored for lipid production in the heterotrophic cultivation. The concentration and the ratio of VFAs exhibited significant influence on cell growth and lipid accumulation. The highest lipid yield coefficient and lipid content of C. protothecoides grown on VFAs were 0.187 g/g and 48.7 %, respectively. The lipid content and fatty acids produced using VFAs as carbon sources were similar to those seen on growth and production using glucose. The techno-economic analysis indicates that the biodiesel derived from the lipids produced by heterotrophic C. protothecoides with VFAs as carbon sources is very promising and competitive with other biofuels and fossil fuels.     

Extracellular release of the glycosylphosphatidylinositol (GPI)-linked Leishmania surface metalloprotease, gp63, is independent of GPI phospholipolysis: implications for parasite virulence.

The major zinc metalloprotease of Leishmania (gp63), an important determinant of parasite virulence, is attached to the parasite surface via a glycosylphosphatidylinositol anchor. Here we report the spontaneous release of proteolytically active gp63 from a number of Leishmania isolates, causing cutaneous and visceral disease. To investigate the mechanism(s) of gp63 release, we transfected a gp63-deficient variant of Leishmania amazonensis with constructs expressing gp63 and various mutants thereof. Surprisingly, approximately half of wild type gp63 was found in the culture supernatant 12 h post-synthesis. Biochemical analysis of the extracellular gp63 revealed two forms of the protein, one that is released from the cell surface, and another, that apparently is directly secreted. Release of cell surface gp63 was significantly reduced when the proteolytic activity of the protein was inactivated by site-specific mutagenesis or inhibited by zinc chelation, suggesting that release involves autoproteolysis. The extracellular gp63 does not contain a glycosylphosphatidylinositol moiety or ethanolamine, indicating that phospholipolysis is not involved in the release process. Release of gp63 is also independent of glycosylation. The finding of proteolytically active, extracellular gp63 produced by multiple Leishmania isolates suggests a potential role of the extracellular enzyme in substrate degradation relevant to their survival in both the mammalian host and the insect vector.     

Effect of fluoride on nucleotides and ribonucleic Acid in germinating corn seedling roots

Investigation was made for the effect of fluoride on plant growth, acid soluble nucleotides, and RNA in germinating corn seedling roots. Fluoride suppresses root growth as measured by changes in fresh weight. Column chromatographic analyses demonstrated that fluoride modifies ratios of acid soluble nucleotide species. The relative amount of nucleotides is altered mainly due to triphosphate nucleotides of which ATP is most accumulated. Paper chromatographic analyses showed that fluoride induces changes of RNA structure. The RNA is characterized by lowered relative content of cytosine and by increased ratio of cytosine to guanine. Adenine is depressed significantly only in the root tissue treated by the highest fluoride concentration.     

Processing of behaviorally relevant temporal parameters of acoustic stimuli by single neurons in the superior olivary nucleus of the leopard frog

Summary Response characteristics of 130 single neurons in the superior olivary nucleus of the northern leopard frog (Rana pipiens pipiens) were examined to determine their selectivity to various behaviorally relevant temporal parameters [rise-fall time, duration, and amplitude modulation (AM) rate of acoustic signals. Response functions were constructed with respect to each of these variables. Neurons with different temporal firing patterns such as tonic, phasic or phasic-burst firing patterns, participated in time domain analysis in specific manners. Phasic neurons manifested preferences for signals with short rise-fall times, thus possessing low-pass response functions with respect to this stimulus parameter; conversely, tonic and phasic-burst units were non-selective and possessed all-pass response functions. A distinction between temporal firing patterns was also observed for duration coding. Whereas phasic units showed no change in the mean spike count with a change in stimulus duration (i.e., all-pass duration response functions), tonic and phasic-burst units gave higher mean spike counts with an increase in stimulus duration (i.e., primary-like high-pass response functions). Phasic units manifested greater response selectivity for AM rate than did tonic or phasic-burst units, and many phasic units were tuned to a narrow range of modulation rates (i.e., band-pass). The results suggest that SON neurons play an important role in the processing of complex acoustic patterns; they perform extensive computations on AM rate as well as other temporal parameters of complex sounds. Moreover, the response selectivities for rise-fall time, duration, and AM rate could often be shown to contribute to the differential responses to complex synthetic and natural sounds.Abbreviations SON superior olivary nucleus - DMN dorsal medullary nucleus - TS torus semicircularis - FTC frequency threshold curve - BF best excitatory frequency - PAM pulsatile amplitude modulation - SAM sinusoidal amplitude modulation - SQAM square-wave amplitude modulation - MTF modulation transfer function - PSTH peri-stimulus time histogram     

Structural analysis of chloroplast tail‐anchored membrane protein recognition by ArsA1

In mammals and yeast, tail‐anchored (TA) membrane proteins destined for the post‐translational pathway are safely delivered to the endoplasmic reticulum (ER) membrane by a well‐known targeting factor, TRC40/Get3. In contrast, the underlying mechanism for translocation of TA proteins in plants remains obscure. How this unique eukaryotic membrane‐trafficking system correctly distinguishes different subsets of TA proteins destined for various organelles, including mitochondria, chloroplasts and the ER, is a key question of long standing. Here, we present crystal structures of algal ArsA1 (the Get3 homolog) in a distinct nucleotide‐free open state and bound to adenylyl‐imidodiphosphate. This approximately 80‐kDa protein possesses a monomeric architecture, with two ATPase domains in a single polypeptide chain. It is capable of binding chloroplast (TOC34 and TOC159) and mitochondrial (TOM7) TA proteins based on features of its transmembrane domain as well as the regions immediately before and after the transmembrane domain. Several helices located above the TA‐binding groove comprise the interlocking hook‐like motif implicated by mutational analyses in TA substrate recognition. Our data provide insights into the molecular basis of the highly specific selectivity of interactions of algal ArsA1 with the correct sets of TA substrates before membrane targeting in plant cells.