Grafted neuronal precursor cells differentiate and integrate in injured hippocampus in experimental pneumococcal meningitis

Bacterial meningitis (BM) frequently causes persisting neurofunctional sequelae. Autopsy studies in patients dying from BM show characteristic apoptotic brain injury to the stem cell niche in the subgranular zone of the hippocampal dentate gyrus (DG), and this form of brain damage is associated with learning and memory deficits in experimental BM. With an eye to potential regenerative therapies, the survival, migration, and differentiation of neuronal precursor cells (NPCs) were evaluated after engraftment into the injured hippocampus in vitro and in vivo in an infant rat model of pneumococcal meningitis. Green fluorescent protein (GFP)-expressing NPCs were grafted into the DG of organotypic hippocampal slice cultures injured by challenge with live Streptococcus pneumoniae. Seven days after engraftment, NPCs had migrated from the site of injection into the injured granular layer of the DG and electro-functionally integrated into the hippocampal network. In vivo, GFP-expressing NPCs migrated within 1 week from the injection site in the hilus region to the injured granular layer of the hippocampal DG and showed neuronal differentiation at 2 and 4 weeks after transplantation. Hippocampal injury induced by BM guides grafted NPCs to the area of brain damage and provides a microenvironment for neuronal differentiation and functional integration.

Protein synthesis in jejunum and liver of neonatal calves fed vitamin A and lactoferrin

Rates of protein synthesis (PS) and turnover are more rapid during the neonatal period than during any other stage of postnatal life. Vitamin A and lactoferrin (Lf) can stimulate PS in neonates. However, newborn calves are vitamin A deficient and have a low Lf status, but plasma vitamin A and Lf levels increase rapidly after ingestion of colostrum. Neonatal calves (n = 6 per group) were fed colostrum or a milk-based formula without or with vitamin A, Lf, or vitamin A plus Lf to study PS in the jejunum and liver. l-[(13)C]Valine was intravenously administered to determine isotopic enrichment of free (nonprotein-bound) Val (AP(Free)) in the protein precursor pool, atom percentage excess (APE) of protein-bound Val, fractional protein synthesis rate (FSR) in the jejunum and liver, and isotopic enrichment of Val in plasma (APE(Pla)) and in the CO(2) of exhaled air (APE(Ex)). The APE, AP(Free), and FSR in the jejunum and liver did not differ significantly among groups. The APE(Ex) increased, whereas APE(Pla) decreased over time, but there were no group differences. Correlations were calculated between FSR(Jej) and histomorphometrical and histochemical data of the jejunum, and between FSR(Liv) and blood metabolites. There were negative correlations between FSR(Liv) and plasma albumin concentrations and between FSR(Jej) and the ratio of villus height:crypt depth, and there was a positive correlation between FSR(Jej) and small intestinal cell proliferation in crypts. Hence, there were no effects of vitamin A and Lf and no interactions between vitamin A and Lf on intestinal and hepatic PS. However, FSR(Jej) was correlated with histomorphometrical traits of the jejunum and FSR(Liv) was correlated with plasma albumin concentrations.

Increase in substance P precursor mRNA in noninflamed small-bowel sections in patients with Crohn's disease

BACKGROUND: Neuropeptides, such as substance P (SP), are mediators of neurogenic inflammation and play an important role in inflammatory disorders. To further investigate the role of the SP pathway in inflammatory bowel disease (IBD), we analyzed the following in normal intestinal tissue specimens and in tissue specimens from patients with Crohn's disease (CD) and ulcerative colitis (UC): neurokinin receptor-1 (NK-1R); its isoforms (NK-1R-L and NK-1R-S); its ligand SP, encoded by preprotachykinin-A (PPT-A); and the SP-degradation enzyme, neutral endopeptidase (NEP). METHODS: Real-time quantitative reverse transcription-polymerase chain reaction was used to simultaneously determine the expression of NK-1R-L, NK-1R-S, and PPT-A. Protein levels of NK-1R and NEP were determined by immunoblot analysis. RESULTS: In noninflamed small-bowel tissue samples of CD patients, PPT-A mRNA expression was significantly increased, whereas there was no difference between inflamed or noninflamed UC and normal intestinal tissue samples. Examining subgroups of diverse intestinal segments from CD and UC samples with various levels of inflammation revealed no differences in NK-1R-L and NK-1R-S mRNA expression, whereas there was a tendency toward overall lower NK-1R-S mRNA copy numbers. Immunoblot analysis showed upregulation of NK-1R protein levels in cases of IBD, with more pronounced enhancement in cases of CD than in UC. For NEP, there were no differences in protein levels in normal, CD, and UC intestinal tissues. COMMENTS: These observations suggest a contribution of SP and its receptor, NK-1R, in the local inflammatory reaction in IBD and particularly in ileal CD. Moreover, significant upregulation of PPT-A mRNA in the noninflamed ileum of these patients suggests an influence of inflamed intestines on their healthy counterparts.

Phosphatidylethanolamine is the precursor of the ethanolamine phosphoglycerol moiety bound to eukaryotic elongation factor 1A

In addition to its conventional role during protein synthesis, eukaryotic elongation factor 1A is involved in other cellular processes. Several regions of interaction between eukaryotic elongation factor 1A and the translational apparatus or the cytoskeleton have been identified, yet the roles of the different post-translational modifications of eukaryotic elongation factor 1A are completely unknown. One amino acid modification, which so far has only been found in eukaryotic elongation factor 1A, consists of ethanolamine-phosphoglycerol attached to two glutamate residues that are conserved between mammals and plants. We now report that ethanolamine-phosphoglycerol is also present in eukaryotic elongation factor 1A of the protozoan parasite Trypanosoma brucei, indicating that this unique protein modification is of ancient origin. In addition, using RNA-mediated gene silencing against enzymes of the Kennedy pathway, we demonstrate that phosphatidylethanolamine is a direct precursor of the ethanolamine-phosphoglycerol moiety. Down-regulation of the expression of ethanolamine kinase and ethanolamine-phosphate cytidylyltransferase results in inhibition of phosphatidylethanolamine synthesis in T. brucei procyclic forms and, concomitantly, in a block in glycosylphosphatidylinositol attachment to procyclins and ethanolamine-phosphoglycerol modification of eukaryotic elongation factor 1A.

Loss of Raf-1 kinase inhibitor protein (RKIP) is strongly associated with high-grade tumor budding and correlates with an aggressive phenotype in pancreatic ductal adenocarcinoma (PDAC)

BACKGROUND Raf-1 kinase inhibitor protein (RKIP) has emerged as a significant metastatic suppressor in a variety of human cancers and is known to inhibit Ras/Raf/MEK/ERK signaling. By suppressing the activation of the NFkB/SNAIL circuit, RKIP can regulate the induction of epithelial-mesenchymal transition (EMT). The aim of this study was to evaluate RKIP expression and to determine its association with clinicopathological features, including EMT in form of tumor budding in pancreatic ductal adenocarcinoma (PDAC). METHODS Staining for RKIP was performed on a multipunch Tissue Microarray (TMA) of 114 well-characterized PDACs with clinico-pathological, follow-up and adjuvant therapy information. RKIP-expression was assessed separately in the main tumor body and in the tumor buds. Another 3 TMAs containing normal pancreatic tissue, precursor lesions (Pancreatic Intraepithelial Neoplasia, PanINs) and matched lymph node metastases were stained in parallel. Cut-off values were calculated by receiver operating characteristic (ROC) curve analysis. RESULTS We found a significant progressive loss of RKIP expression between normal pancreatic ductal epithelia (average: 74%), precursor lesions (PanINs; average: 37%), PDAC (average 20%) and lymph node metastases (average 8%, p<0.0001). RKIP expression was significantly lower in tumor buds (average: 6%) compared to the main tumor body (average 20%; p<0.005). RKIP loss in the tumor body was marginally associated with advanced T-stage (p=0.0599) as well as high-grade peritumoral (p=0.0048) and intratumoral budding (p=0.0373). RKIP loss in the buds showed a clear association with advanced T stage (p=0.0089). CONCLUSIONS The progressive loss of RKIP seems to play a major role in the neoplastic transformation of pancreas, correlates with aggressive features in PDAC and is associated with the presence of EMT in form of tumor budding.

Wingless activity in the precursor cells specifies neuronal migratory behavior in the Drosophila nerve cord.

Neurons and their precursor cells are formed in different regions within the developing CNS, but they migrate and occupy very specific sites in the mature CNS. The ultimate position of neurons is crucial for establishing proper synaptic connectivity in the brain. In Drosophila, despite its extensive use as a model system to study neurogenesis, we know almost nothing about neuronal migration or its regulation. In this paper, I show that one of the most studied neuronal pairs in the Drosophila nerve cord, RP2/sib, has a complicated migratory route. Based on my studies on Wingless (Wg) signaling, I report that the neuronal migratory pattern is determined at the precursor cell stage level. The results show that Wg activity in the precursor neuroectodermal and neuroblast levels specify neuronal migratory pattern two divisions later, thus, well ahead of the actual migratory event. Moreover, at least two downstream genes, Cut and Zfh1, are involved in this process but their role is at the downstream neuronal level. The functional importance of normal neuronal migration and the requirement of Wg signaling for the process are indicated by the finding that mislocated RP2 neurons in embryos mutant for Wg-signaling fail to properly send out their axon projection.

Eliciting the mitochondrial unfolded protein response via NAD(+) repletion reverses fatty liver disease

With no approved pharmacological treatment, non-alcoholic fatty liver disease (NAFLD) is now the most common cause of chronic liver disease in western countries and its worldwide prevalence continues to increase along with the growing obesity epidemic. Here we show that a high-fat high-sucrose (HFHS) diet, eliciting chronic hepatosteatosis resembling human fatty liver, lowers hepatic NAD(+) levels driving reductions in hepatic mitochondrial content, function and ATP levels, in conjunction with robust increases in hepatic weight, lipid content and peroxidation in C57BL/6J mice. In an effort to assess the effect of NAD(+) repletion on the development of steatosis in mice, nicotinamide riboside (NR), a precursor for NAD(+) biosynthesis, was given to mice concomitant, as preventive strategy (NR-Prev), and as a therapeutic intervention (NR-Ther), to a HFHS diet. We demonstrate that NR prevents and reverts NAFLD by inducing a SIRT1- and SIRT3-dependent mitochondrial unfolded protein response (UPR(mt) ), triggering an adaptive mitohormetic pathway to increase hepatic β-oxidation and mitochondrial complex content and activity. The cell-autonomous beneficial component of NR treatment was revealed in liver-specific Sirt1 KO mice (Sirt1(hep-/-) ), while Apolipoprotein E-deficient (Apoe(-/-) ) mice challenged with a high-fat high-cholesterol diet (HFC), affirmed the use of NR in other independent models of NAFLD. CONCLUSION Our data warrant the future evaluation of NAD(+) boosting strategies to manage the development or progression of NAFLD. This article is protected by copyright. All rights reserved.

The Outer Chloroplast Envelope Protein OEP16-1 for Plastid Import of NADPH:Protochlorophyllide Oxidoreductase A in Arabidopsis thaliana

The outer plastid envelope protein OEP16-1 was previously identified as an amino acid-selective channel protein and translocation pore for NADPH:protochlorophyllide oxidoreductase A (PORA). Reverse genetic approaches used to dissect these mutually not exclusive functions of OEP16-1 in planta have led to descriptions of different phenotypes resulting from the presence of several mutant lines in the SALK_024018 seed stock. In addition to the T-DNA insertion in the AtOEP16-1 gene, lines were purified that contain two additional T-DNA insertions and as yet unidentified point mutations. In a first attempt to resolve the genetic basis of four different lines in the SALK_024018 seed stock, we used genetic transformation with the OEP16-1 cDNA and segregation analyses after crossing out presumed point mutations. We show that AtOEP16-1 is involved in PORA precursor import and by virtue of this activity confers photoprotection onto etiolated seedlings during greening

Purification and Characterization of AsES Protein A Subtilisin Secreted by Acremonium Strictum is a Novel Plant Defense Elicitor.

In this work, the purification and characterization of an extracellular elicitor protein, designated AsES, produced by an avirulent isolate of the strawberry pathogen Acremonium strictum, are reported. The defense eliciting activity present in culture filtrates was recovered and purified by ultrafiltration (cutoff, 30 kDa), anionic exchange (Q-Sepharose, pH 7.5), and hydrophobic interaction (phenyl-Sepharose) chromatographies. Two-dimensional SDS-PAGE of the purified active fraction revealed a single spot of 34 kDa and pI 8.8. HPLC (C2/C18) and MS/MS analysis confirmed purification to homogeneity. Foliar spray with AsES provided a total systemic protection against anthracnose disease in strawberry, accompanied by the expression of defense-related genes (i.e. PR1 and Chi2-1). Accumulation of reactive oxygen species (e.g. H2O2 and O2̇̄) and callose was also observed in Arabidopsis. By using degenerate primers designed from the partial amino acid sequences and rapid amplification reactions of cDNA ends, the complete AsES-coding cDNA of 1167 nucleotides was obtained. The deduced amino acid sequence showed significant identity with fungal serine proteinases of the subtilisin family, indicating that AsES is synthesized as a larger precursor containing a 15-residue secretory signal peptide and a 90-residue peptidase inhibitor I9 domain in addition to the 283-residue mature protein. AsES exhibited proteolytic activity in vitro, and its resistance eliciting activity was eliminated when inhibited with PMSF, suggesting that its proteolytic activity is required to induce the defense response. This is, to our knowledge, the first report of a fungal subtilisin that shows eliciting activity in plants. This finding could contribute to develop disease biocontrol strategies in plants by activating its innate immunity.

Regulation of chloroplast protein import through a protochlorophyllide-responsive transit peptide

NADPH:protochlorophyllide (Pchlide) oxidoreductase (POR) is the key enzyme of chlorophyll biosynthesis in angiosperms. In barley, two POR enzymes, termed PORA and PORB, exist. Both are nucleus-encoded plastid proteins that must be imported posttranslationally from the cytosol. Whereas the import of the precursor of PORA, pPORA, previously has been shown to depend on Pchlide, the import of pPORB occurred constitutively. To study this striking difference, chimeric precursor proteins were constructed in which the transit sequences of the pPORA and pPORB were exchanged and fused to either their cognate polypeptides or to a cytosolic dihydrofolate reductase (DHFR) reporter protein of mouse. As shown here, the transit peptide of the pPORA (transA) conferred the Pchlide requirement of import onto both the mature PORB and the DHFR. By contrast, the transit peptide of the pPORB directed the reporter protein into both chloroplasts that contained or lacked translocation-active Pchlide. In vitro binding studies further demonstrated that the transit peptide of the pPORA, but not of the pPORB, is able to bind Pchlide. We conclude that the import of the authentic pPORA and that of the transA-PORB and transA-DHFR fusion proteins is regulated by a direct transit peptide-Pchlide interaction, which is likely to occur in the plastid envelope, a major site of porphyrin biosynthesis.

Cell death induction by the acute promyelocytic leukemia-specific PML/RARα fusion protein

PML/RARα is the abnormal protein product generated by the acute promyelocytic leukemia-specific t(15;17). Expression of PML/RARα in hematopoietic precursor cell lines induces block of differentiation and promotes survival. We report here that PML/RARα has a potent growth inhibitory effect on all nonhematopoietic cell lines and on the majority of the hematopoietic cell lines tested. Inducible expression of PML/RARα in fibroblasts demonstrated that the basis for the growth suppression is induction of cell death. Deletion of relevant promyelocytic leukemia (PML) and retinoic acid receptor (RARα) domains within the fusion protein revealed that its growth inhibitory effect depends on the integrity of the PML aminoterminal region (RING, B1, B2, and coiled coil regions) and the RARα DNA binding region. Analysis of the nuclear localization of the same PML/RARα deletion mutants by immunofluorescence and cell fractionation revealed that the biological activity of the fusion protein correlates with its microspeckled localization and its association to the nuclear matrix. The PML aminoterminal region, but not the RARα zinc fingers, is required for the proper nuclear localization of PML/RARα. We propose that the matrix-associated microspeckles are the active sites of PML/RARα and that targeting of RARα sequences to this specific nuclear subdomain through PML sequences is crucial to the activity of the fusion protein on survival regulation.

Influenza C virus CM2 integral membrane glycoprotein is produced from a polypeptide precursor by cleavage of an internal signal sequence

The influenza C virus CM2 protein is a small glycosylated integral membrane protein (115 residues) that spans the membrane once and contains a cleavable signal sequence at its N terminus. The coding region for CM2 (CM2 ORF) is located at the C terminus of the 342-amino acid (aa) ORF of a colinear mRNA transcript derived from influenza C virus RNA segment 6. Splicing of the colinear transcript introduces a translational stop codon into the ORF and the spliced mRNA encodes the viral matrix protein (CM1) (242 aa). The mechanism of CM2 translation was investigated by using in vitro and in vivo translation of RNA transcripts. It was found that the colinear mRNA derived from influenza C virus RNA segment 6 serves as the mRNA for CM2. Furthermore, CM2 translation does not depend on any of the three in-frame methionine residues located at the beginning of CM2 ORF. Rather, CM2 is a proteolytic cleavage product of the p42 protein product encoded by the colinear mRNA: a cleavage event that involves the recognition and cleavage of an internal signal peptide presumably by signal peptidase resident in the endoplasmic reticulum. Alteration of the predicted signal peptidase cleavage site by mutagenesis blocked generation of CM2. The other polypeptide species resulting from the cleavage of p42, designated p31, contains the CM1 coding region and an additional C-terminal 17 aa (formerly the CM2 signal peptide). Protein p31, in comparison to CM1, displays characteristics of an integral membrane protein.

The dimensions of the protein import channels in the outer and inner mitochondrial membranes

Most mitochondrial proteins are imported into mitochondria through transmembrane channels composed largely, and perhaps exclusively, of proteins. We have determined the effective internal diameter of the protein import channel in the mitochondrial outer membrane to be between 20 Å and 26 Å during translocation. The diameter of the import channel in the inner membrane is smaller than the diameter of the outer membrane import channel. These results were obtained by measuring the effect of rigid steric bulk introduced into precursor proteins on import.

Identification of a thiamin-dependent synthase in Escherichia coli required for the formation of the 1-deoxy-d-xylulose 5-phosphate precursor to isoprenoids, thiamin, and pyridoxol

In Escherichia coli, 1-deoxy-d-xylulose (or its 5-phosphate, DXP) is the biosynthetic precursor to isopentenyl diphosphate [Broers, S. T. J. (1994) Dissertation (Eidgenössische Technische Hochschule, Zürich)], thiamin, and pyridoxol [Himmeldirk, K., Kennedy, I. A., Hill, R. E., Sayer, B. G. & Spenser, I. D. (1996) Chem. Commun. 1187–1188]. Here we show that an open reading frame at 9 min on the chromosomal map of E. coli encodes an enzyme (deoxyxylulose-5-phosphate synthase, DXP synthase) that catalyzes a thiamin diphosphate-dependent acyloin condensation reaction between C atoms 2 and 3 of pyruvate and glyceraldehyde 3-phosphate to yield DXP. We have cloned and overexpressed the gene (dxs), and the enzyme was purified 17-fold to a specific activity of 0.85 unit/mg of protein. The reaction catalyzed by DXP synthase yielded exclusively DXP, which was characterized by 1H and 31P NMR spectroscopy. Although DXP synthase of E. coli shows sequence similarity to both transketolases and the E1 subunit of pyruvate dehydrogenase, it is a member of a distinct protein family, and putative DXP synthase sequences appear to be widespread in bacteria and plant chloroplasts.

The N terminus of the Drosophila Numb protein directs membrane association and actin-dependent asymmetric localization

Drosophila Numb is a membrane associated protein of 557 amino acids (aa) that localizes asymmetrically into a cortical crescent in mitotic neural precursor cells and segregates into one of the daughter cells, where it is required for correct cell fate specification. We demonstrate here that asymmetric localization but not membrane localization of Numb in Drosophila embryos is inhibited by latrunculin A, an inhibitor of actin assembly. We also show that deletion of either the first 41 aa or aa 41–118 of Numb eliminates both localization to the cell membrane and asymmetric localization during mitosis, whereas C-terminal deletions or deletions of central portions of Numb do not affect its subcellular localization. Fusion of the first 76 or the first 119 aa of Numb to β-galactosidase results in a fusion protein that localizes to the cell membrane, but fails to localize asymmetrically during mitosis. In contrast, a fusion protein containing the first 227 aa of Numb and β-galactosidase localizes asymmetrically during mitosis and segregates into the same daughter cell as the endogenous Numb protein, demonstrating that the first 227 aa of the Numb protein are sufficient for asymmetric localization.