Proteins that mediate protein aggregation and cytotoxicity distinguish Alzheimer's hippocampus from normal controls

Neurodegenerative diseases are distinguished by characteristic protein aggregates initiated by disease‐specific ‘seed’ proteins; however, roles of other co‐aggregated proteins remain largely unexplored. Compact hippocampal aggregates were purified from Alzheimer's and control‐subject pools using magnetic‐bead immunoaffinity pulldowns. Their components were fractionated by electrophoretic mobility and analyzed by high‐resolution proteomics. Although total detergent‐insoluble aggregates from Alzheimer's and controls had similar protein content, within the fractions isolated by tau or Aβ_1–42 pulldown, the protein constituents of Alzheimer‐derived aggregates were more abundant, diverse, and post‐translationally modified than those from controls. Tau‐ and Aβ‐containing aggregates were distinguished by multiple components, and yet shared >90% of their protein constituents, implying similar accretion mechanisms. Alzheimer‐specific protein enrichment in tau‐containing aggregates was corroborated for individuals by three analyses. Five proteins inferred to co‐aggregate with tau were confirmed by precise in situ methods, including proximity ligation amplification that requires co‐localization within 40 nm. Nematode orthologs of 21 proteins, which showed Alzheimer‐specific enrichment in tau‐containing aggregates, were assessed for aggregation‐promoting roles in C. elegans by RNA‐interference ‘knockdown’. Fifteen knockdowns (71%) rescued paralysis of worms expressing muscle Aβ, and 12 (57%) rescued chemotaxis disrupted by neuronal Aβ expression. Proteins identified in compact human aggregates, bound by antibody to total tau, were thus shown to play causal roles in aggregation based on nematode models triggered by Aβ_1–42. These observations imply shared mechanisms driving both types of aggregation, and/or aggregate‐mediated cross‐talk between tau and Aβ. Knowledge of protein components that promote protein accrual in diverse aggregate types implicates common mechanisms and identifies novel targets for drug intervention.     

Serum apolipoproteins C-I and C-III are reduced in stomach cancer patients: results from MALDI-based peptidome and immuno-based clinical assays

Finding new peptide biomarkers for stomach cancer in human sera that can be implemented into a clinically practicable prediction method for monitoring of stomach cancer. We studied the serum peptidome from two different biorepositories. We first employed a C8-reverse phase liquid chromatography approach for sample purification, followed by mass-spectrometry analysis. These were applied onto serum samples from cancer-free controls and stomach cancer patients at various clinical stages. We then created a bioinformatics analysis pipeline and identified peptide signature discriminating stomach adenocarcinoma patients from cancer-free controls. Matrix Assisted Laser Desorption/Ionization-Time of Flight (MALDI-TOF) results from 103 samples revealed 9 signature peptides; with prediction accuracy of 89% in the training set and 88% in the validation set. Three of the discriminating peptides discovered were fragments of Apolipoproteins C-I and C-III (apoC-I and C-III); we further quantified their serum levels, as well as CA19-9 and CRP, employing quantitative commercial-clinical assays in 142 samples. ApoC-I and apoC-III quantitative results correlated with the MS results. We then employed apoB-100-normalized apoC-I and apoC-III, CA19-9 and CRP levels to generate rules set for stomach cancer prediction. For training, we used sera from one repository, and for validation, we used sera from the second repository. Prediction accuracies of 88.4% and 74.4% were obtained in the training and validation sets, respectively. Serum levels of apoC-I and apoC-III combined with other clinical parameters can serve as a basis for the formulation of a diagnostic score for stomach cancer patients.     

Non-small cell lung carcinoma cell motility, rac activation and metastatic dissemination are mediated by protein kinase C epsilon

Background

Protein kinase C (PKC) ε, a key signaling transducer implicated in mitogenesis, survival, and cancer progression, is overexpressed in human primary non-small cell lung cancer (NSCLC). The role of PKCε in lung cancer metastasis has not yet been established.

Principal Findings

Here we show that RNAi-mediated knockdown of PKCε in H358, H1299, H322, and A549 NSCLC impairs activation of the small GTPase Rac1 in response to phorbol 12-myristate 13-acetate (PMA), serum, or epidermal growth factor (EGF). PKCε depletion markedly impaired the ability of NSCLC cells to form membrane ruffles and migrate. Similar results were observed by pharmacological inhibition of PKCε with εV1-2, a specific PKCε inhibitor. PKCε was also required for invasiveness of NSCLC cells and modulated the secretion of extracellular matrix proteases and protease inhibitors. Finally, we found that PKCε-depleted NSCLC cells fail to disseminate to lungs in a mouse model of metastasis.

Conclusions

Our results implicate PKCε as a key mediator of Rac signaling and motility of lung cancer cells, highlighting its potential as a therapeutic target.     

VapC toxins from Mycobacterium tuberculosis are ribonucleases that differentially inhibit growth and are neutralized by cognate VapB antitoxins

The chromosome of Mycobacterium tuberculosis (Mtb) encodes forty seven toxin-antitoxin modules belonging to the VapBC family. The role of these modules in the physiology of Mtb and the function(s) served by their expansion are unknown. We investigated ten vapBC modules from Mtb and the single vapBC from M. smegmatis. Of the Mtb vapCs assessed, only Rv0549c, Rv0595c, Rv2549c and Rv2829c were toxic when expressed from a tetracycline-regulated promoter in M. smegmatis. The same genes displayed toxicity when conditionally expressed in Mtb. Toxicity of Rv2549c in M. smegmatis correlated with the level of protein expressed, suggesting that the VapC level must exceed a threshold for toxicity to be observed. In addition, the level of Rv2456 protein induced in M. smegmatis was markedly lower than Rv2549c, which may account for the lack of toxicity of this and other VapCs scored as 'non-toxic'. The growth inhibitory effects of toxic VapCs were neutralized by expression of the cognate VapB as part of a vapBC operon or from a different chromosomal locus, while that of non-cognate antitoxins did not. These results demonstrated a specificity of interaction between VapCs and their cognate VapBs, a finding corroborated by yeast two-hybrid analyses. Deletion of selected vapC or vapBC genes did not affect mycobacterial growth in vitro, but rendered the organisms more susceptible to growth inhibition following toxic VapC expression. However, toxicity of 'non-toxic' VapCs was not unveiled in deletion mutant strains, even when the mutation eliminated the corresponding cognate VapB, presumably due to insufficient levels of VapC protein. Together with the ribonuclease (RNase) activity demonstrated for Rv0065 and Rv0617--VapC proteins with similarity to Rv0549c and Rv3320c, respectively--these results suggest that the VapBC family potentially provides an abundant source of RNase activity in Mtb, which may profoundly impact the physiology of the organism.     

Differential DNA methylation with age displays both common and dynamic features across human tissues that are influenced by CpG landscape

Background

DNA methylation is an epigenetic modification that changes with age in human tissues, although the mechanisms and specificity of this process are still poorly understood. We compared CpG methylation changes with age across 283 human blood, brain, kidney, and skeletal muscle samples using methylation arrays to identify tissue-specific age effects.

Results

We found age-associated CpGs (ageCGs) that are both tissue-specific and common across tissues. Tissue-specific ageCGs are frequently located outside CpG islands with decreased methylation, and common ageCGs show the opposite trend. AgeCGs are significantly associated with poorly expressed genes, but those with decreasing methylation are linked with higher tissue-specific expression levels compared with increasing methylation. Therefore, tissue-specific gene expression may protect against common age-dependent methylation. Distinguished from other tissues, skeletal muscle ageCGs are more associated with expression, enriched near genes related to myofiber contraction, and closer to muscle-specific CTCF binding sites. Kidney-specific ageCGs are more increasingly methylated compared to other tissues as measured by affiliation with kidney-specific expressed genes. Underlying chromatin features also mark common and tissue-specific age effects reflective of poised and active chromatin states, respectively. In contrast with decreasingly methylated ageCGs, increasingly methylated ageCGs are also generally further from CTCF binding sites and enriched within lamina associated domains.

Conclusions

Our data identified common and tissue-specific DNA methylation changes with age that are reflective of CpG landscape and suggests both common and unique alterations within human tissues. Our findings also indicate that a simple epigenetic drift model is insufficient to explain all age-related changes in DNA methylation.     

Isolation and characterization of phosphorylated oligosaccharides from alpha-N-acetylglucosaminidase that are recognized by cell-surface receptors.

Adsorptive endocytosis of lysosomal enzymes by fibroblasts and hepatocytes involves binding to cell surface receptors that recognize on lysosomal enzymes a phosphorylated carbohydrate, most likely a mannose 6-phosphate residue [Kaplan et al. (1977) Proc. Natl Acad. Sci. U.S.A. 74, 2026-2030; Ullrich et al. (1978) Hoppe-Seyler's Z. Physiol. Chem. 359, 1591-1598]. Loss of alpha-N-acetylglucosaminidase endocytosis after treatment with endoglucosaminidase H indicated that the recognition site of alpha-N-acetylglucosaminidase is located on N-glycosidically linked oligosaccharides of the high mannose type. Acidic oligosaccharides with an average molecular weight of 2200 were liberated from alpha-N-acetylglucosaminidase by endoglucosaminidase H. These oligosaccharides were susceptible to degradation by alkaline phosphatase, alpha-mannosidase and beta-N-acetylglucosaminidase. At the non-reducing terminal these oligosaccharides bear phosphorylated mannose and/or N-acetylglucosamine residues.     

Early and late periodic patterns of even skipped expression are controlled by distinct regulatory elements that respond to different spatial cues

We have identified the regulatory sequences required for the periodic expression of the Drosophila pair rule gene even skipped (eve). We find that the gradually changing pattern of periodic eve expression during early embryogenesis is directed by two distinct regulatory programs. Initially, eve expression in individual stripes is established by different regulatory elements, each of which responds to nonperiodic spatial cues provided, at least in part, by the gap genes. Later, coordinate expression of eve in all seven stripes is directed by a single regulatory region that responds to periodic cues provided by primary pair rule genes, including eve itself. As a consequence of this two-step regulatory program, eve functions both in the establishment of the periodic pattern of gene expression and in the subsequent specification of parasegmental boundaries.     

MicroRNA-99a-3p/GRP94 axis affects metastatic progression of human papillary thyroid carcinoma by regulating ITGA2 expression and localization

Papillary thyroid cancer (PTC) usually has favorable prognosis;however,distant metastasis is a leading cause of death associated with PTC.MicroRNA-99a-3p (miR-9...     

Simultaneous transfer of tumorigenic and metastatic phenotypes by transfection with genomic DNA from a human cutaneous squamous cell carcinoma

High-molecular-weight genomic DNA isolated from a human cutaneous squamous cell carcinoma (AS) was assayed for its ability to induce tumorigenic transformation of NIH 3T3 cells. Subcutaneous injection of NIH 3T3 cells cotransfected with DNAs from AS tumor and pSV2-neo plasmid not only induced tumors at the site of injection, but also metastasized spontaneously to the lungs in 100% of nude mice injected. DNA isolated from a representative primary tumor and a metastasis was again used in a second round of transfection. Injection of secondary transfectants into nude mice again resulted in induction of both subcutaneous tumors and spontaneous long metastases. Southern blot hybridization with ras-specific probes revealed that DNA from both primary tumors and metastases induced by AS tumor DNA contained highly amplified Ha-ras oncogene. Furthermore, DNAs from secondary tumors and metastases induced by DNA from a primary tumor and a metastasis also contained similar highly amplified Ha-ras oncogene. These results suggest that the amplified Ha-ras oncogene may be responsible for induction of both tumorigenic and metastatic phenotypes in NIH 3T3 cells transfected with DNA from AS tumor.     

Galectin-3 binds lactosaminylated lipooligosaccharides from Neisseria gonorrhoeae and is selectively expressed by mucosal epithelial cells that are infected

Galectins are a family of beta-galactoside binding proteins that have been proposed as host receptors for bacteria because beta-galactoside carbohydrates are common in bacterial membrane glycolipid lipooligosaccharides (LOS) and lipopolysaccharides. We investigated the interaction of galectin-3 with gonococcal LOS that make lactosyl (Lc2 or Lac), paraglobosyl (nLc4; LNnT; lacto-N-neotetraose), gangliosyl (IV3GalNAcnLc4), and neolactohexaosyl (nLc6, lactonorhexaosyl) oligosaccharides. All but gangliosyl LOS terminate in beta-galactoside. Galectin-3 had the highest affinity for the nLc6 LOS, which is made by a strain that is highly infectious for the male urethra, but also bound nLc4 LOS and to a Lac LOS. The lacto-N-neotetraose tetrasaccharide was a more potent inhibitor of galectin-3 binding to LOS than either lactose or N-acetyllactosamine. The relative affinity of galectin-3 for gonococci mirrored its affinity for purified LOS. Western blot analysis revealed expression of galectin-3 by human endometrial adenocarcinoma and prostatic epithelial cells that can be invaded by gonococci. Immunohistochemistry of human fallopian tube epithelium showed localized expression of galectin-3 by non-ciliated cells, the specific cell gonococci invade in this tissue. We conclude that because of its location and affinity for gonococcal LOS galectin-3 could play a role in gonococcal infection.     

Release of DEFB126 from macaque sperm and completion of capacitation are triggered by conditions that simulate periovulatory oviductal fluid

Capacitation of macaque sperm in vitro has been achieved efficiently only with the addition of both cyclic nucleotides and methylxanthines. The use of these exogenous sperm activators clouds an understanding of the normal mechanisms underlying capacitation and may slow early embryo development following in vitro fertilization (IVF). We demonstrate that culture medium which simulates periovulatory oviductal fluid with respect to bicarbonate (HCO(3)(-)) and glucose concentration induces capacitation in a high percentage of macaque sperm as determined by the ability of sperm to undergo both the release of coating protein DEFB126 and the zona pellucida-induced acrosome reaction (AR). Few sperm were able to undergo the AR following 6 hr incubation in medium containing either 35 mM HCO(3)(-) (approximately 7.2 pH) or 90 mM HCO(3)(-) (approximately pH 7.8) with 5 mM glucose. When glucose concentration was lowered to 0.5 mM to match levels reported for women at midcycle, the AR rate increased significantly in sperm incubated in both levels of HCO(3)(-), indicating that glucose interferes with sperm responsiveness to increasing HCO(3)(-) concentration observed in the primate oviduct during ovulation. Even greater synchronization of capacitation could be achieved with nonphysiologic extremes of alkalinity or energy substrate deprivation. In the latter case, sperm achieved high rates of IVF. A shift in pH from 7.2 to 7.8 in a HEPES-buffered medium was sufficient to remove DEFB126 from the surface of most sperm after only 3 hr. The loss of DEFB126 from sperm under periovulaory fluid conditions has implications for the timing of release of sperm from the oviductal reservoir.     

Cytidines in tRNAs that are required intact by ATP/CTP:tRNA nucleotidyltransferases from Escherichia coli and Saccharomyces cerevisiae.

Individual species of tRNA from Escherichia coli were treated with hydrazine/3 M NaCl to modify cytidine residues. The chemically modified tRNAs were used as substrate for ATP/CTP: tRNA nucleotidyltransferases from E. coli and yeast, with [alpha-32P]ATP as cosubstrate. tRNAs that were labeled were analyzed for their content of modified cytidines. Cytidines at positions 74 and 75 were found to be required chemically intact for interaction with both enzymes. C56 was also required intact by the E. coli enzyme in all tRNAs, and by the yeast enzyme in several instances. C61 was found to be important in seven of 14 tRNAs with the E. coli enzyme but only in four of 13 tRNAs with that from yeast. Our results support a model in which nucleotidyltransferase extends from the 3' end of its tRNA substrate across the top of the stacked array of bases in the accepter- and psi-stems to the corner of the molecule where the D- and psi-loops are juxtaposed.     

Increase in cytoplasmic Ca2+ and stimulation of calcitonin secretion from human medullary thyroid carcinoma cells by the gastrin-releasing peptide.

Examination was made of the effects of gastrin-releasing peptide (GRP) on human medullary thyroid carcinoma cells (TT cells). GRP stimulated calcitonin(CT) release in a concentration-dependent manner at 0.1-1000 nmol/l. On adding forskolin along with GRP, CT release was greater than by GRP alone. The stimulatory effect of A23187 was not additive. Intracellular free calcium concentration ([Ca2+]i) was measured for individual TT cells loaded with fura-2. The addition of GRP caused a rapid and transient rise in [Ca2+]i in a concentration-dependent manner followed by a sustained increase in [Ca2+]i. In the medium without Ca2+, this sustained increase did not occur and the concentration of CT release from TT cells by GRP was reduced by approximately a half. GRP would thus appear to be importantly involved in the regulation of thyroid C cell function through modulation of [Ca2+]i.     

Establishment of a CSF-producing cell line from a human gastric carcinoma and characteristics of the CSF produced by that cell line

A human cell line producing colony-stimulating factor has been established in vitro from a human gastric carcinoma. The cell line was transplantable into nude mice which developed a marked neutrophilia. The cell line has been maintained for three years. The cells grew in a monolayered sheet and produced colony-stimulating factors that enhanced the formation of granulocyte and monocyte colonies in vitro with mouse bone marrow cells as the target and granulocyte colonies with human bone marrow cells as the target.     

Caterpillar color patterns are determined by a two‐phase melanin gene prepatterning process: new evidence from tan and laccase2

SUMMARY The larval color patterns in Lepidoptera exhibit splendid diversity, and identifying the genes responsible for pigment distribution is essential to understanding color‐pattern evolution. The swallowtail butterfly, Papilio xuthus, is a good candidate for analyzing marking‐associated genes because its body markings change dramatically at the final molt. Moreover, the silkworm Bombyx mori is most suitable for identification of lab‐generated color mutants because genome information and many color mutants are available. Here, we analyzed the expression pattern of 10 melanin‐related genes in P. xuthus, and analyzed whether these genes were responsible for Bombyx larval color mutants. We found that seven genes correlated strongly with the stage‐specific larval cuticular markings of P. xuthus, suggesting that, compared with Drosophila, more genes showed marking specificity in lepidopteran larvae. We newly found that the expression of both tan and laccase2 is strongly correlated with the larval black markings in both P. xuthus and B. mori. The results of F2 linkage analysis and mutant analysis strongly suggest that tan is the responsible gene for Bombyx larval color mutant rouge, and that tan is important in emphasizing black markings of lepidopteran larvae. Detailed comparison of temporal and spatial expression patterns showed that larval cuticular markings were regulated at two different phases. Marking‐specific expression of oxidizing enzymes preceded the marking‐specific expression of melanin synthesis enzymes at mRNA level, which is the reverse of the melanin synthesis step.     

Phosphatidate phosphatase from Saccharomyces cerevisiae. Isolation of 45- and 104-kDa forms of the enzyme that are differentially regulated by inositol

Immunoblot analysis of cell extracts using antibodies specific for the 91-kDa form of membrane-associated phosphatidate phosphatase from Saccharomyces cerevisiae (Lin, Y.-P., and Carman, G.M. (1989) J. Biol. Chem. 264, 8641-8645) revealed the existence of a 45-kDa form of the enzyme. Immunoblot analysis also showed that the 91-kDa form of the enzyme was a proteolytic product of a 104-kDa enzyme. The mitochondrial fraction contained the 45-kDa enzyme, whereas the microsomal fraction contained the 45- and 104-kDa enzymes. In vivo labeling experiments showed that the 104-kDa form of phosphatidate phosphatase was not a precursor of the 45-kDa form of the enzyme. The 45- and 104-kDa forms of phosphatidate phosphatase were purified and characterized. The enzymological properties of both enzymes were similar. However, the phosphatidate phosphatase 45- and 104-kDa proteins differed with respect to their isoelectric points and peptide fragments resulting from V8 proteolysis and cyanogen bromide cleavage. The expression of the phosphatidate phosphatase 45- and 104-kDa enzymes were regulated differentially in cells supplemented with inositol. The addition of inositol to the growth medium resulted in the induction of the phosphatidate phosphatase 45-kDa enzyme. The expression of the 104-kDa enzyme was not affected by inositol. Both forms of phosphatidate phosphatase were induced when cells entered the stationary phase of growth.     

Molecular cloning and heterologous expression of novel glucosyltransferases from tobacco cultured cells that have broad substrate specificity and are induced by salicylic acid and auxin.

Scopoletin is one of the phytoalexins in tobacco. Cells of the T-13 cell line (Nicotiana tabacum L. Bright Yellow) accumulate a large amount of scopoletin, also known as 7-hydroxy-6-methoxycoumarin, as a glucoconjugate, scopolin, in vacuoles. We report here the molecular cloning of glucosyltransferases that can catalyze the glucosylation of many kinds of secondary metabolites including scopoletin. Two cDNAs encoding glucosyltransferase (NtGT1a and NtGT1b) were isolated from a cDNA library derived from the tobacco T-13 cell line by screening with heterologous cDNAs as a probe. The deduced amino-acid sequences of NtGT1a and NtGT1b exhibited 92% identity with each other, approximately 20-50% identities with other reported glucosyltransferases. Heterologous expression of these genes in Escherichia coli showed that the recombinant enzymes had glucosylation activity against both flavonoids and coumarins. They also strongly reacted with 2-naphthol as a substrate. These recombinant enzymes can utilize UDP-glucose as the sugar donor, but they can also utilize UDP-xylose as a weak donor. RNA blot analysis showed that these genes are induced by salicylic acid and auxin, but the time course of the expression was different. This result is similar to the changes in scopoletin glucosylation activity in these tobacco cells after addition of these plant growth regulators. These results might suggest that one of the roles of the products of these genes is scopoletin glucosylation, in response to salicylic acid and/or auxin, together with the other glucosyltransferases in tobacco cells.