A therapeutic peptide in lupus alters autophagic processes and stability of MHCII molecules in MRL/lpr B cells

The P140 phosphopeptide encompassing residues 131-151 of the spliceosomal U1-70K snRNP protein displays protective properties in lupus patients and MRL/lpr mice. It increases peripheral blood lymphocyte apoptosis via a mechanism involving γδ T cells. After intravenous administration, P140 accumulates in the lungs and spleen. It binds both the HSC70/Hsp73 chaperone and MHC class II (MHCII) molecules, which colocalize in splenic MRL/lpr B cells. Expression of HSC70 and MHCII, which is increased in MRL/lpr splenic B cells, is diminished after P140 administration. P140 impairs refolding properties of HSC70 and alters expression of stable MHCII molecules in B lymphocytes. In MRL/lpr B cells, P140 increases the accumulation of the autophagy markers p62/SQSTM1 and LC3-II, consistent with a downregulation of autophagic flux. Our study reveals a very unique property of P140 peptide that alters the autophagy pathway leading to a defect of endogenous (auto)antigen processing in MRL/lpr antigen-presenting B cells and a decrease of T cell priming and signaling.     

Potent Antiproliferative Cembrenoids Accumulate in Tobacco upon Infection with Rhodococcus fascians and Trigger Unusual Microtubule Dynamics in Human Glioblastoma Cells

Aims

Though plant metabolic changes are known to occur during interactions with bacteria, these were rarely challenged for pharmacologically active compounds suitable for further drug development. Here, the occurrence of specific chemicals with antiproliferative activity against human cancer cell lines was evidenced in hyperplasia (leafy galls) induced when plants interact with particular phytopathogens, such as the Actinomycete Rhodococcus fascians.

Methods

We examined leafy galls fraction F3.1.1 on cell proliferation, cell division and cytoskeletal disorganization of human cancer cell lines using time-lapse videomicroscopy imaging, combined with flow cytometry and immunofluorescence analysis. We determined the F3.1.1-fraction composition by gas chromatography coupled to mass spectrometry.

Results

The leafy galls induced on tobacco by R. fascians yielded fraction F3.1.1 which inhibited proliferation of glioblastoma U373 cells with an IC₅₀ of 4.5 µg/mL, F.3.1.1 was shown to increase cell division duration, cause nuclear morphological deformations and cell enlargement, and, at higher concentrations, karyokinesis defects leading to polyploidization and apoptosis. F3.1.1 consisted of a mixture of isomers belonging to the cembrenoids. The cellular defects induced by F3.1.1 were caused by a peculiar cytoskeletal disorganization, with the occurrence of fragmented tubulin and strongly organized microtubule aggregates within the same cell. Colchicine, paclitaxel, and cembrene also affected U373 cell proliferation and karyokinesis, but the induced microtubule rearrangement was very different from that provoked by F3.1.1. Altogether our data indicate that the cembrenoid isomers in F3.1.1 have a unique mode of action and are able to simultaneously modulate microtubule polymerization and stability.     

A recurrent deletion of DPY19L2 causes infertility in man by blocking sperm head elongation and acrosome formation

An increasing number of couples require medical assistance to achieve a pregnancy, and more than 2% of the births in Western countries now result from assisted reproductive technologies. To identify genetic variants responsible for male infertility, we performed a whole-genome SNP scan on patients presenting with total globozoospermia, a primary infertility phenotype characterized by the presence of 100% round acrosomeless spermatozoa in the ejaculate. This strategy allowed us to identify in most patients (15/20) a 200 kb homozygous deletion encompassing only DPY19L2, which is highly expressed in the testis. Although there was no known function for DPY19L2 in humans, previous work indicated that its ortholog in C. elegans is involved in cell polarity. In man, the DPY19L2 region has been described as a copy-number variant (CNV) found to be duplicated and heterozygously deleted in healthy individuals. We show here that the breakpoints of the deletions are located on a highly homologous 28 kb low copy repeat (LCR) sequence present on each side of DPY19L2, indicating that the identified deletions were probably produced by nonallelic homologous recombination (NAHR) between these two regions. We demonstrate that patients with globozoospermia have a homozygous deletion of DPY19L2, thus indicating that DPY19L2 is necessary in men for sperm head elongation and acrosome formation. A molecular diagnosis can now be proposed to affected men; the presence of the deletion confirms the diagnosis of globozoospermia and assigns a poor prognosis for the success of in vitro fertilization.     

The H3 histone chaperone NASPSIM3 escorts CenH3 in Arabidopsis

Centromeres define the chromosomal position where kinetochores form to link the chromosome to microtubules during mitosis and meiosis. Centromere identity is determined by incorporation of a specific histone H3 variant termed CenH3. As for other histones, escort and deposition of CenH3 must be ensured by histone chaperones, which handle the non‐nucleosomal CenH3 pool and replenish CenH3 chromatin in dividing cells. Here, we show that the Arabidopsis orthologue of the mammalian NUCLEAR AUTOANTIGENIC SPERM PROTEIN (NASP) and Schizosaccharomyces pombe histone chaperone Sim3 is a soluble nuclear protein that binds the histone variant CenH3 and affects its abundance at the centromeres. NASP^SIM3 is co‐expressed with Arabidopsis CenH3 in dividing cells and binds directly to both the N‐terminal tail and the histone fold domain of non‐nucleosomal CenH3. Reduced NASP^SIM3 expression negatively affects CenH3 deposition, identifying NASP^SIM3 as a CenH3 histone chaperone.     

Antiviral Action of Methylated β-Lactoglobulin on the Human Influenza Virus A Subtype H3N2

Antiviral activity of methylated β-lactoglobulin (Met-BLG) against H3N2 infected into MDCK cell lines depended on concentration of Met-BLG, viral load, and duration of infection. IC_50% of the hemagglutination activity for 1 and 0.2 MOI (multiplicity of infection) after 24 h of incubation at 37 °C in the presence of 5% CO₂ were 20 ± 0.8 and 17 ± 0.7 μg mL^?1 Met-BLG, respectively. Longer incubation period (4 days) was associated with low IC_50% of the hemagglutination activity (7.1 ± 0.3 μg mL^?1 Met-BLG) and low IC_50% of immuno-fluorescence of viral nucleoproteins (9.7 ± 0.4 μg mL^?1 Met-BLG) when using 0.2 and 0.1 MOI, respectively. A concentration of 25 μg mL^?1 of Met-BLG reduced the amount of replicating virus by about 2 and 1.3 logs when the viral load was 0.01 and 0.1 MOI, respectively, while higher concentrations reduced it by about 5–6 logs. Antiviral action of Met-BLG was coupled with a cellular protective action, which reached 100% when using 0.01 and 0.1 MOI and 83% when using 1.0 MOI. The time of Met-BLG addition after the viral infection was determinant for its antiviral efficacy and for its protection of the infected MDCK cell lines. Anti-hemagglutination action and cell protective action decreased gradually and in parallel with the delay in the time of Met-BLG addition to disappear totally after 10 h delay.     

Synthesis and biological evaluation of (3,4,5-trimethoxyphenyl)indol-3-ylmethane derivatives as potential antivascular agents

Combretastatin A-4 (CSA-4), a stilbene derivative, is a potent vascular disrupting agent (VDA) with the structural requirement of a cis-configuration to maintain a molecular geometry and a correct orientation of both phenyl groups. A series of indolic analogues of CSA-4 was synthesized by means of an efficient strategy. Six compounds (20b, 25b-27b, 32b, and 35b) were identified as potent inhibitors of tubulin polymerization and also displayed cytotoxic activities on B16 melanoma cells at a nanomolar level. Both activities were well correlated with the ability to induce morphological changes of EA.hy 926 endothelial cells. In conclusion, the cis-stilbene skeleton of CSA-4 could conveniently be replaced by the 3-aroylindolic moiety, thus avoiding any isomerization leading to inactive trans compounds.     

Similar protein phosphatases control starch metabolism in plants and glycogen metabolism in mammals

We report that protein phosphorylation is involved in the control of starch metabolism in Arabidopsis leaves at night. sex4 (starch excess 4) mutants, which have strongly reduced rates of starch metabolism, lack a protein predicted to be a dual specificity protein phosphatase. We have shown that this protein is chloroplastic and can bind to glucans and have presented evidence that it acts to regulate the initial steps of starch degradation at the granule surface. Remarkably, the most closely related protein to SEX4 outside the plant kingdom is laforin, a glucan-binding protein phosphatase required for the metabolism of the mammalian storage carbohydrate glycogen and implicated in a severe form of epilepsy (Lafora disease) in humans.     

Brain magnetic resonance study of Mecp2 deletion effects on anatomy and metabolism

Rett syndrome, a neurodevelopmental X-linked disorder, represents the most important genetic cause of severe mental retardation in the female population and results from a mutation in the gene encoding methyl-CpG-binding protein 2 (MECP2). We report here the first characterization of Mecp2-null mice, by in vivo magnetic resonance imaging and spectroscopy, delineating the cerebral phenotype associated with the lack of Mecp2. We performed a morphometric study that revealed a size reduction of the whole brain and of structures involved in cognitive and motor functions (cerebellum and motor cortex). Significant metabolic anomalies, including reduced N-acetylaspartate, myo-inositol, and glutamine plus glutamate, and increased choline levels were evidenced. These findings indicate that not only neuronal but also glial metabolism is affected in Mecp2-null mice. Furthermore, we uncovered an important reduction of brain ATP level, a hitherto undetected anomaly of energy metabolism that may reflect and contribute to cerebral injury and dysfunction.