Heparanase expression in invasive trophoblasts and acute vascular damage

Heparan sulfate proteoglycans play a pivotal role in tissue function, development, inflammation, and immunity. We have identified a novel cDNA encoding human heparanase, an enzyme thought to cleave heparan sulfate in physiology and disease, and have located the HEP gene on human chromosome 4q21. Monoclonal antibodies against human heparanase located the enzyme along invasive extravillous trophoblasts of human placenta and along endothelial cells in organ xenografts targeted by hyperacute rejection, both sites of heparan sulfate digestion. Heparanase deposition was evident in arterial walls in normal tissues; however, vascular heparan sulfate cleavage was coincident with heparanase enzyme during inflammatory episodes. These findings suggest that heparanase elaboration and control of catalytic activity may contribute to the development and pathogenesis of vascular disease and suggest that heparanase intervention might be a useful therapeutic target.     

Identification of microRNAs as potential prognostic markers in ependymoma

Introduction

We have examined expression of microRNAs (miRNAs) in ependymomas to identify molecular markers of value for clinical management. miRNAs are non-coding RNAs that can block mRNA translation and affect mRNA stability. Changes in the expression of miRNAs have been correlated with many human cancers.

Materials and Methods

We have utilized TaqMan Low Density Arrays to evaluate the expression of 365 miRNAs in ependymomas and normal brain tissue. We first demonstrated the similarity of expression profiles of paired frozen tissue (FT) and paraffin-embedded specimens (FFPE). We compared the miRNA expression profiles of 34 FFPE ependymoma samples with 8 microdissected normal brain tissue specimens enriched for ependymal cells. miRNA expression profiles were then correlated with tumor location, histology and other clinicopathological features.

Results

We have identified miRNAs that are over-expressed in ependymomas, such as miR-135a and miR-17-5p, and down-regulated, such as miR-383 and miR-485-5p. We have also uncovered associations between expression of specific miRNAs which portend a worse prognosis. For example, we have identified a cluster of miRNAs on human chromosome 14q32 that is associated with time to relapse. We also found that miR-203 is an independent marker for relapse compared to the parameters that are currently used. Additionally, we have identified three miRNAs (let-7d, miR-596 and miR-367) that strongly correlate to overall survival.

Conclusion

We have identified miRNAs that are differentially expressed in ependymomas compared with normal ependymal tissue. We have also uncovered significant associations of miRNAs with clinical behavior. This is the first report of clinically relevant miRNAs in ependymomas.     

Cloning and expression of a new member of prolactin-related protein in bovine placenta: bovine prolactin-related protein-VII

This study reports the identification and sequence of a full-length cDNA for a new member of bovine prolactin-related protein (bPRP-VII) and its quantitative and localized expression in the placenta. A full-length bPRP-VII cDNA was cloned with a 929-nucleotide open-reading-frame corresponding to a protein of 238 amino acids. The predicted amino acid sequence shares 63% homology with bPRP-I and 70% with bPRP-VI. bPRP-VII has eight cysteine residues with four disulfide bonds, which is more abundant than that of other bPRPs. RT-PCR detected bPRP-VII only in the placenta. In the placenta, mRNA was expressed in the cotyledon and intercotyledonary tissues throughout gestation. Quantitative real-time RT-PCR analysis exhibited a high expression of bPRP-VII mRNA in the fetal membrane at Day 27 of gestation. In the placentome on Day 60 of gestation, in situ hybridization analysis evidenced bPRP-VII mRNA in binucleate cells. bPRP-VII gene produced a mature protein in mammalian cell expression system. Approximately 29kDa protein was confirmed in this by the Western blot analysis with FLAG epitope tag. Expression profiles and localization were similar to those of bPRP-I. Although the functional data remain to be examined, a new member of the bPRP-VII gene was cloned. In addition to bPRP-I, bPRP-VII may take on an important functional role in implantation.     

Cloning and expression of two new prolactin-related proteins,prolactin-related protein-VIII and -IX,in bovine placenta

Background  

Prolactin-related proteins (PRPs) are specific proteins of the growth hormone/prolactin (GH/PRL) family in bovine placenta. This study reports the identification and sequencing of a full-length cDNA for two new members of bovine PRPs, bPRP-VIII and -IX, and their localization and quantitative expression in bovine placenta.     

Identification of COS markers in the Pinaceae

Conserved ortholog set (COS) markers are evolutionary conserved, single-copy genes, identified from large databases of express sequence tags (ESTs). They are of particular use for constructing syntenic genetic maps among species. In this study, we identified a set of 1,813 putative single-copy COS markers between spruce and loblolly pine, then designed primers for 931 of these markers and tested these primers with DNA from spruce, pine, and Douglas fir. Of these 931 primers, 56% (524) amplified a product in both spruce and pine, and 71% (373) of these were single-banded; 224 amplicons were single-banded in all three species. Even though these COS markers were selected from large EST databases, a substantial proportion (20–30%) of amplicons displayed multiple bands or smears, suggesting significant paralogy. Sequencing of three single-banded amplicons showed high nucleotide similarities among 29 conifer species, suggesting orthology of single-banded amplicons. Screening for COS marker polymorphism in two pedigrees of white spruce and two pedigrees of loblolly pine revealed an average informativeness of 36% for spruce and 24% for pine (e.g., at least one parent was heterozygous for a single-nucleotide polymorphism within the entire amplified product). This corresponds to an average nucleotide heterozygosity of 0.05% and 0.03%, respectively, which is considerably lower than that found in other studies of spruce and pine. Thus, the advantages of COS markers for constructing syntenic maps are offset by their lower polymorphism. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Identification of phosphoproteins as possible differentiation markers in all-trans-retinoic acid-treated neuroblastoma cells

Background

Neuroblastic tumors account for 9–10% of pediatric tumors and neuroblastoma (NB) is the first cause of death in pre-school age children. NB is classified in four stages, depending on the extent of spreading. A fifth type of NB, so-called stage 4S (S for special), includes patients with metastatic tumors but with an overall survival that approximates 75% at five years. In most of these cases, the tumor regresses spontaneously and regression is probably associated with delayed neuroblast cell differentiation.

Methodology/Principal Findings

In order to identify new early markers to follow and predict this process for diagnostic and therapeutics intents, we mimicked the differentiation process treating NB cell line SJ-NK-P with all-trans-retinoic acid (ATRA) at different times; therefore the cell proteomic pattern by mass spectrometry and the phosphoproteomic pattern by a 2-DE approach coupled with anti-phosphoserine and anti-phosphotyrosine western blotting were studied.

Conclusions/Significance

Proteomic analysis identified only two proteins whose expression was significantly different in treated cells versus control cells: nucleoside diphosphate kinase A (NDKA) and reticulocalbin-1 (RCN1), which were both downregulated after 9 days of ATRA treatment. However, phosphoproteomic analysis identified 8 proteins that were differentially serine-phosphorylated and 3 that were differentially tyrosine-phosphorylated after ATRA treatment. All proteins were significantly regulated (at least 0.5-fold down-regulated). Our results suggest that differentially phosphorylated proteins could be considered as more promising markers of differentiation for NB than differentially expressed proteins.     

Identification of a complex of the three forms of the rat liver asialoglycoprotein receptor

We have generated antibodies against synthetic peptides which represent the carboxyl terminus of either the major, or the two minor, forms of the rat hepatic lectin which recognizes galactose-terminated glycoproteins (asialoglycoproteins). The antibodies were shown to be specific for the form of the lectin containing the immunizing peptide sequence by the following: reaction with purified lectin after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoprecipitation of sodium dodecyl sulfate-denatured lectin, immunoprecipitation of lectin synthesized in vitro. These antibodies, however, precipitated all three rat hepatic lectin forms from nonionic detergent extracts of hepatocytes labeled with 125I via the lactoperoxidase catalyzed technique. A similar result was obtained if antibody was bound to intact cells prior to solubilization with detergent and collection of the immune complexes. We conclude that at least the plasma membrane-associated fraction of the rat hepatic lectin forms exists as a heterotypic complex.     

Identification of serum sirtuins as novel noninvasive protein markers for frailty

Frailty has emerged as a major health issue among older patients. A consensus on definition and diagnosis is yet to be achieved. Various biochemical abnormalities have been reported in frailty. Activation of sirtuins, a conserved family of NAD‐dependent proteins, is one of the many mimics of calorie restriction which improves lifespan and health in experimental animals. In this cross‐sectional study, we assessed the circulating sirtuin levels in 119 (59.5%) nonfrail and 81 (40.5%) frail individuals, diagnosed by Fried's criteria. Serum SIRT1, SIRT2, and SIRT3 were estimated by surface plasmon resonance (SPR) and Western blot. Serum sirtuins level in mean+SD; SIRT1 (nonfrail –4.67 ± 0.48 ng/μL; frail – 3.72 ± 0.48 ng/μL; P < 0.0001), SIRT2 (nonfrail – 15.18 ± 2.94 ng/μL; frail – 14.19 ± 2.66 ng/μL; P = 0.016), and SIRT3 (nonfrail‐7.72 ± 1.84 ng/μL; frail – 6.12 ± 0.97 ng/μL; P < 0.0001) levels were significantly lower among frail patients compared with the nonfrail. In multivariable regression analysis, lower sirtuins level were significantly associated with frailty after adjusting age, gender, diabetes mellitus, hypertension, cognitive status (Mini Mental State Examination scores) and number of comorbidities. For detecting the optimum diagnostic cutoff value a ROC analysis was carried out. The area under curve for SIRT1 was 0.9037 (cutoff – 4.29 ng/μL; sensitivity – 81.48%; specificity – 79.83%) and SIRT3 was 0.7988 (cutoff – 6.61 ng/μL; sensitivity – 70.37%; specificity – 70.59%). This study shows that lower circulating SIRT1 and SIRT3 levels can be distinctive marker of frailty.     

Secretory proteins as markers for cellular phenotypes in rat salivary glands

The neonatal submandibular glands (SMG) of the rat contain two types of cells: Type III cells secrete a group of proteins in response to beta-adrenergic stimulation, and Type I cells secrete a different protein, called Protein C (89 kDa), in response to cholinergic stimuli (Ball and Redman, 1984). Polyclonal antibodies raised to Protein B1 (26 kDa) showed that the several proteins in the B1-Immunoreactive Protein (B1-IP) group are localized exclusively to Type III cells. Although we expected that antibodies to Protein B1 would label only the submandibular gland, we found instead that the serous demilunes of the sublingual gland (SLG) and the acinar cells and intercalated ducts of the parotid gland (PRG) were strongly reactive in both the neonate and the adult. Immunoelectrophoretic analysis of gland extracts showed the major reactive species in the sublingual gland to have different mobilities than the B1-IP. On the other hand, reactive species in the parotid gland had mobilities identical to those of two SMG proteins. In the adult SMG, the neonatal Type I and Type III cells are not present, and the acinar cells are devoid of B1-IP reactivity; however, the cells of the intercalated ducts have components reactive with anti-B1 antibodies, and these do not appear to be identical to any neonatal bands. In contrast to the submandibular gland, the adult parotid and sublingual glands retain the localization of B1-IP reactivity in PRG acinar and intercalated duct cells and in SLG demilunes, and they show the neonatal immunoelectrophoretic pattern. This raises the possibility that the major B1-IP species in the adult PRG may be identical to transient proteins of the neonatal SMG.     

Identification of microsatellite markers in the rye genome

The rye genomic library, which consists of DNA fragments in the range of 0.5–1.1 kb, was screened for the presence of tri-and tetranucleotide and compound microsatellites. Of the 1,600,000 clones analysed, 102 clones were positive and 41 were suitable for SSR primer pair design. Twenty-six primer pairs amplified specific products, and six of them were capable of detecting polymorphism among 30 rye accessions of different genetic backgrounds. Using a set of Chinese Spring-Imperial wheat-rye addition lines, it was possible to locate 3 newly identified microsatellites on chromosomes 3R, 4R and 7R.     

Identification of three isozyme proteins of the catalytic subunit of the Na,K-ATPase in rat brain

Molecular genetic evidence indicates that there should be three different (Na+ + K+)-stimulated ATPase (Na,K-ATPase) alpha subunit isozymes in the brain where previously only two ("alpha" and "alpha(+)") were resolved as proteins. To detect and identify alpha 1, alpha 2, and alpha 3 isozymes, polypeptides made by cell-free translation (Schneider, J.W., Mercer, R.W., Gilmore-Hebert, M., Utset, M.F., Lai, C., Greene, A., and Benz, E.J., Jr. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 284-288) were analyzed by gel electrophoresis and proteolytic fingerprinting. Synthetic alpha 1 comigrated with tissue alpha 1, while alpha 2 and alpha 3 comigrated with the leading and trailing edges, respectively, of tissue "alpha(+)." Proteolytic fingerprints of newborn rat brain Na,K-ATPase labeled in vivo with L-[35S]methionine indicated the presence of alpha 1 and alpha 3, and a low level of alpha 2. Monoclonal antibodies were characterized by the electrophoretic mobility of their antigens and by their ability to recognize the Na,K-ATPases of kidney, brain, and skeletal muscle. The antibodies were used to assess isozyme expression in the brain. All three isozymes increased in abundance during development from the 18-day fetus to the adult. Small changes were seen in the relative level of expression of alpha 1 and alpha 3 at different developmental ages, while alpha 2 expression increased markedly between the neonate and adult. In adult brain, all three isozymes were found in all brain regions examined. We conclude that all three isozymes are expressed as proteins and that their expression and distribution must be under complex control. No single developmental age or macroscopic brain region provides an exclusive source of any of the isozymes.     

CD44 adhesion molecule and neuro-glial proteoglycan NG2 as invasive markers of glioma

Glioma invasion into the CNS involves the interaction of tumor cells with the host’s cells and extracellular matrix (ECM) molecules. In this study, the expression of ECM-associated and cell-associated proteins such as the transmembrane CD44 adhesion molecule and neuro-glial proteoglycan 2 (NG2), a member of the chondroitin sulfate proteoglycan family, were evaluated during glioma progression, in vitro and in vivo, using a model of a highly invasive and aggressive intracerebral mouse G-26 glioma. We found a marked increase in CD44 and NG2 expression in brain tissue containing glioma. The glioma levels of these proteins gradually increased over time to reach 3–15 times the levels in the contralateral control. NG2 and CD44 expression paralleled progression of the glioma, being higher on days 14 and 21 than on day 2 post-glioma implant. In addition, when invading glioma crossed the midline in the advanced tumor stage, levels of each of these proteins in the contralateral tissue were elevated, but were still significantly lower than in the ipsilateral, tumor-bearing hemisphere. Immunohistochemistry of advanced stage G-26 glioma (day 21) showed CD44 expression to be most prominent at the front of the glioma invasion line, sharply separated from normal brain parenchyma which expressed glial fibrillary acidic protein (GFAP). However, single CD44 positive cells that escaped the tumor mass penetrated between the astrocytes that encased the tumor at its periphery. In contrast, NG2 was expressed on nearly all glioma cells within the tumor mass but less so at the leading edge of the tumor. The NG2 positive cells were clearly demarcated and morphologically distinguishable from GFAP positive cells and only sporadic, small groups of NG2 positive cells were seen in the GFAP positive zone of the neuropil. Taken together, these data show that during glioma progression in the brain, the level and pattern of glioma-associated molecules such as CD44 and NG2 may aid in tracing and targeting the invading glioma cells.     

Identification,expression, and tissue distribution of the three rat lysyl hydroxylase isoforms

Lysyl hydroxylases (LH) (procollagen-lysine 2-oxoglutarate 5-dioxygenase; PLOD) catalyse the hydroxylation of lysine residues during the post-translational modification of collagenous proteins. In this paper, we describe the first identification and cloning of LH isoforms 2 and 3 from the rat, including both LH2 splice variants (LH2a and LH2b). The rat LHs are expressed in almost all tissue and cell types examined, indicating a probable lack of tissue specificity for LH function. All LH isoforms were stably transfected into CHO-K1 cells and this represents the first example of recombinant LH production in a eukaryotic cell line. Expression and production of all LH isoforms led to an increase in total collagen synthesis. LH1 and LH2a expression and production led to an increase in total pyridinium cross-link production. Evidence that LH2a possesses telopeptide lysyl hydroxylase activity, previously thought to be a novel enzyme, is presented.     

Identification of three protein kinases which phosphorylate threonyl-tRNA synthetase from rat liver

Threonyl-tRNA synthetase is phosphorylated in Chinese hamster ovary cells labeled with 32Pi [(1984) J. Biol. Chem. 259, 11160-11161]. Phosphorylation of the purified synthetase from rat liver has been examined with five different protein kinases. Three of the enzymes phosphorylate the synthetase, protease activated kinase I, the cAMP-dependent protein kinase, and the Ca2+, phospholipid-dependent protein kinase. Phosphorylation occurs exclusively on seryl residues. Two-dimensional phosphopeptide maps of tryptic digests of the phosphorylated synthetase are distinct with each protein kinase. These data suggest that multiple phosphorylation of the synthetase may occur in vivo.     

Identification of cytoskeletal markers for the different microvilli and cell types of the rat vomeronasal sensory epithelium

The vomeronasal organ (VNO) of the mammal nose is specialized to detect pheromones. The presumed site of the chemosensory signal transduction of pheromones is the vomeronasal brush border of the VNO sensory epithelium, which has been shown to contain two different sets of microvilli: (i) the tall microvilli of supporting cells and (ii) the short microvilli of the chemoreceptive VNO neurons that branch and intermingle with the basal portions of the longer supporting cell microvilli. A key problem when studying the subcellular distribution of possible VNO signal transduction molecules at the light microscope level is the clear discrimination of immunosignals derived from dendritic microvilli of the VNO neurons and surrounding supporting cell structures. In the present study we therefore looked for cytoskeletal marker proteins, that might help to distinguish at the light microscope level between the two sets of microvilli. By immunostaining we found that the VNO dendritic microvilli can be selectively labelled with antibodies to the calcium-sensitive actin filament-bundling protein villin, whereas supporting cell microvilli contain the actin filament cross-linking protein fimbrin, but not villin. Useful cytoplasmic marker molecules for cellular discrimination were cytokeratin 18 for supporting cells and β-tubulin for dendrites of VNO neurons. A further finding was that the non-sensory epithelium of the rat VNO contains brush cells, a cell type that appears to be involved in certain aspects of chemoreception in the gut. Brush cells or other structures of the vomeronasal brush border did not contain α-gustducin.     

Identification of three high molecular mass cysteine proteinases from rat skeletal muscle

Three cysteine proteinases were isolated from the post-myofibrillar fraction of rat skeletal muscle. Proteinase I preferentially hydrolyzes Z-Phe—Arg-NMec with pH optimum at 8–9. The enzyme activity is stabilized by ATP against thermal inactivation. Proteinase II and III were not resolved by anion-exchange chromatography, by affinity chromatography on Arginine—Sepharose or by gel filtration. Proteinase II, splitting Bz-Val---Gly---Arg-NMec optimally at pH 10–10.5, is inactivated by ATP, whereas Proteinase III, hydrolyzing Suc-Ala---Ala---Phe-NMec at pH 7–7.5 is not affected by the nucleotide. The molecular mass of proteinase I is about 750 000 and that of proteinase II and III is about 650 000, as determined by gel filtration.