Alpha-enolase Research Articles

Proteomic analysis of the reproductive tract fluids from tropically-adapted Santa Ines rams

The present study is focused on the proteome of reproductive tract fluids from tropically-adapted Santa Ines rams. Seminal plasma, cauda epididymal (CEF) and vesicular gland fluid (VGF) proteins were analyzed by 2-D electrophoresis and mass spectrometry. Seminal plasma maps contained 302 ± 16 spots, within the 4-7 pH range. From these maps, 73 spots were identified, corresponding to 41 proteins. Ram Seminal Vesicle Proteins (RSVP) 14 and 22kDa and bodhesins 1 and 2 represented the most abundant seminal components. Other seminal proteins included clusterin, angiotensin-converting enzyme, matrix metalloproteinase-2, tissue-inhibitor of metalloproteinase-2, plasma glutamate carboxypeptidase, albumin, lactoferrin, alpha enolase, peroxiredoxin, leucine aminopeptidase, β-galactosidase, among others. Later, seminal plasma gels were run within narrow pH intervals (3.9-5.1; 4.7-5.9; 5.5-6.7), allowing the additional identification of 21 proteins not detected in 4-7 pH maps. Major proteins of CEF and VGF were albumin and transferrin, and RSVPs, respectively. Western blots confirmed that RSVPs were mainly present in VGF while bodhesins, in VGF and CEF. Based on RT-PCR, RSVP and bodhesin genes were primarily expressed in the vesicular glands. In summary, the reproductive tract fluids of Brazilian hairy rams contain several categories of proteins, with potential roles in sperm protection, capacitation, acrosome reaction and sperm-oocyte interaction.

Anti-Endothelial Cell Antibodies in Patients with Alzheimer Disease (P05.052)

Objective: To examine the presence of antiendothelial cell antibodies (AECAs) specific for Alzheimer disease (AD), and to evaluate the association of these antibodies with cognitive impairment. Background Previous studies have reported that the BBB impairment may be a pathogenetic mechanism contributing to cognitive impairment in AD. However, the mechanism of BBB impairment in AD is unknown. We hypothesized that AECA associated with BBB impairment is present in AD. Design/Methods: The study included AD patients (aged ≥ 60 years; 24 patients), control subjects without neurologic diseases (aged ≥ 60 years; 19 subjects), healthy control subjects (aged Results: We detected 5 spots that reacted with AECAs, which frequently appeared in sera from AD patients. These 5 immunoreactive spots were identified as Vimentin; Protein disulfide-isomerase A3; Alpha enolase; Mitochondrial import receptor subunit TOM40 homolog (Tom40); and Annexin A2. The anti-Tom40 antibody was identified significantly more frequently in AD patients than it was in control subjects or patients with multiple sclerosis. In AD patients, the mean scores for the mini-mental state examination were significantly lower for patients who were positive for the anti-Tom40 antibody than they were for those patients who were negative for the anti-Tom40 antibody. Conclusions: The anti-Tom40 antibody is significantly associated with cognitive impairment in AD patients. Disclosure: Dr. Kimura has nothing to disclose. Dr. Sakurai has nothing to disclose. Dr. Yamada has nothing to disclose. Dr. Koumura has nothing to disclose. Dr. Hayashi has nothing to disclose. Dr. Tanaka has nothing to disclose. Dr. Hozumi has nothing to disclose. Dr. Otaki has nothing to disclose. Dr. Chousa has nothing to disclose. Dr. Inuzuka has nothing to disclose.

Intravitreal triamcinolone for cancer-associated retinopathy refractory to systemic therapy

PurposeThe purpose of this study is to report the use of intravitreal triamcinolone for treatment of cancer-associated retinopathy (CAR) refractory to systemic therapy.MethodsThis was a retrospective chart review study.ResultsA 67-year-old man presented with cancer-associated retinopathy with antibodies against a 46-kDa retinal protein, alpha enolase. There was disease progression despite therapy with mycophenolate and intravenous immunoglobulin. Serial intravitreal injections of triamcinolone resulted in restoration of photoreceptor anatomy on optical coherence tomography and visual improvement. The patient’s vision was preserved at 20/40 OD and 20/32 OS until his death from lung cancer 31 months after CAR diagnosis.ConclusionsIntravitreal triamcinolone may be beneficial for maintenance of vision in patients with CAR.

Molecular modelling, docking and interaction studies of human-plasmogen and salmonella enolase with enolase inhibitors.

Salmonella enteric serovar Typhi Ty2 is a human specific pathogen and an etiological agent for typhoid fever. Most of Salmonella serotypes produce glycogen which has a comparatively minor role in virulence and colonization, but has a more significant role in survival. Enzymes present in glycolytic pathway of bacteria help bacteria to survive by activating other factors inside host. Numerous pathogenic bacteria species intervene with the plasminogen system, and this plasminogen–enolase association may play a critical role in the virulence of S. Typhi by causing direct damage to the host cell extracellular matrix, possibly by enzymic degradation of extracellular matrix proteins or other protein constituents. In this study, molecular modelling of enolase of Salmonella has been accomplished in silico by comparative modelling; we have then analyzed Human alpha enolase which is a homodimer and serves on epithelial cells with our model. Both Structures were docked by D-tartronate semialdehyde phosphate (TSP) and 3-aminoenolpyruvate phosphate (AEP) enolase inhibitors. Our study shows that salmonella enolase and human enolase have different active sites in their structure. This will help in development of new ligands, more suitable for inhibiting bacterial survival inside host as vaccines for typhoid fever are not fully protective. The study also confirmed that enolase Salmonella and Human Plasminogen suggested direct physical interaction between both of them as the activation loop of plasminogen residues showed conformational changes similar to the tissue type plasminogen activator. Various computational biology tools were used for our present study such as Modeller, Molegro Virtual Docker, Grommacs.

The lectin Helix pomatia agglutinin recognizes O-GlcNAc containing glycoproteins in human breast cancer

There has been considerable interest in understanding the epitopes that bind the lectin Helix pomatia agglutinin (HPA) in breast cancer as the lectin has been shown to identify glycosylation changes associated with the development of metastatic disease. HPA has previously been shown to recognize aberrant O-linked α-N-acetylgalactosamine (GalNAcα)/mucin glycosylation in cancer, including exposed Tn epitopes. However, recent glycan-array analysis reported that diverse epitopes are also recognized by the lectin, e.g. consortium for functional glycomics (CFG) data: GalNAcα1,3Gal; β-GalNAc; GlcNAcβ1,4Gal. The intriguing observations from the CFG array led to this study, in which HPA-binding epitopes were localized and characterized in an in vitro model of breast cancer metastasis. HMT3522 (benign disease), BT474 (primary cancer) and T47D/MCF7 (metastatic cancer) cells were assessed in confocal microscopy-based co-localization studies and a glycoproteomic analysis based on 2-dimensional electrophoresis (2DE), western blotting and mass spectrometry was adopted. HPA binding correlated with levels of integrin α6, transcription factors heterogeneous nuclear ribonuclear protein (HnRNP) H1, HnRNP D-like, HnRNP A2/B1 as well as heat shock protein 27 (Hsp27), glial fibrillary acidic protein and enolase 1 (ENO1). These glycoproteins were non-detectable in the non-metastatic breast cancer cell lines. The recognition of HnRNPs, Hsp27 and ENO1 by HPA correlated with O-GlcNAcylation of these proteins. Integrin α6 was the most abundant HPA glycoprotein in the breast cancer cells with a metastatic phenotype; this concurred with previous findings in colorectal cancer. This is the first report in which HPA has been shown to bind O-GlcNAcylated transcription factors. This class of proteins represents a new means by which HPA differentiates cancer cells with an aggressive metastatic phenotype.

Effects of Space Flight on the Expression of Liver Proteins in the Mouse

Raw data derived from mass spectroscopic (MS) analyses of formalin-fixed paraffin-embedded (FFPE) tissue sections of the essential metabolic organ, liver, allocated by the provider (Amgen) from mice subjected to 13 days of microgravity on NASA Flight STS-118 were analyzed by two different search engines, using shotgun proteomics. With the eight statistically significant readouts in hand, Ingenuity Pathway Analysis (IPA) was employed to visualize probable biologic pathway relationships among proteins that might be associated with alterations in liver biochemistry due to space flight. Most noteworthy was the finding of up-regulation of the first urea cycle enzyme carbamoylphosphate synthetase, consistent with increased amino acid catabolism resulting from gravitational changes, and/ or other stress associated with missions in space. Down-regulation of fructose-bisphosphate aldolase B, regucalcin, ribonuclease UK114, alpha enolase, glycine N-methyltransferase and S-adenosyl methionine synthetase isoform type-1 was observed. 60 kDa heat shock protein was elevated.

P1-01-16: Detecting a Breast Carcinoma-Deriving B-Cell Response: An Immunoproteomics Biomarker Approach.

Abstract Background: Noninvasive biomarkers for the early detection of breast cancer are crucial due to the fact that the relapse risk of breast cancer is rising with the time point of its detection. Currently, none of the reported molecular biomarkers is established for the clinical use as a diagnostic tool. Previous proteomics-based studies showed the immunogenicity of breast carcinoma and the following B-cell mediated immune response. As a result, several autoantibodies against tumor proteins were detected in the sera of breast cancer patients. However, these putative biomarkers are still lacking of clinically reliable specificity and sensitivity, even of better discrimination of cancer patients when combining different biomarkers. The search for a new antibody biomarker signature remains very important as a potential cancer detection tool. For further investigations we analyzed the antibody pattern in serum samples of diseased patients and healthy controls after incubation with whole protein extract from a native carcinoma and identified the putative tumor-specific immunoreactive antigens. Materials and methods: For our de novo profiling of tumor antigens we used a protein extract from a primary invasive ductal carcinoma. Sera from 20 women, of which 19 were diagnosed with breast carcinoma and one with DCIS (CA), and 20 sera from age-matched healthy donors (CTRL) were obtained and pooled separately. For an optimal separation of tumor antigens a two-dimensional sodium dodecylsulfate gel electrophoresis (2D SDS-PAGE) was applied. Following immunoblots with each serum pool were performed and the immunospecific reactions visualized with a horseradish peroxidase-conjugated anti-IgG antibody. The relevant tumor antigens were identified via Matrix-Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI TOF-TOF MS). Results: After the incubation of each serum pool with tumor antigens we obtained different extensive antibody profiles, whereas the visualized immunoreactive components varied in the signal intensity. We identified over 10 tumor antigens which reacted with the corresponding autoantibodies in CA or CTRL approach, showing again the complexity of immune response. Besides of already described breast carcinoma related antigens like alpha enolase, which showed immune reaction also with the healthy serum pool, we identified several potential antigens of interest like peroxiredoxin 6 which showed a strong immune response only after the incubation with cancer sera. Discussion: In our study we visualized a complex immune response pattern showing the autoantibody profiles in cancer and healthy sera against tumor-deriving antigens. Also some of identified breast carcinoma antigens were already described; other novel breast carcinoma-related antigens were detected too. Our next step in the intriguing search for cancer antibody biomarkers is the individual screening of sera to confirm the specificity of the tumor-deriving B-cell responses and the validation of these results by using an independent study population. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-01-16.

Age-dependent changes in the proteome following complete spinal cord transection in a postnatal South American opossum (Monodelphis domestica).

Recovery from severe spinal injury in adults is limited, compared to immature animals who demonstrate some capacity for repair. Using laboratory opossums (Monodelphis domestica), the aim was to compare proteomic responses to injury at two ages: one when there is axonal growth across the lesion and substantial behavioural recovery and one when no axonal growth occurs. Anaesthetized pups at postnatal day (P) 7 or P28 were subjected to complete transection of the spinal cord at thoracic level T10. Cords were collected 1 or 7 days after injury and from age-matched controls. Proteins were separated based on isoelectric point and subunit molecular weight; those whose expression levels changed following injury were identified by densitometry and analysed by mass spectrometry. Fifty-six unique proteins were identified as differentially regulated in response to spinal transection at both ages combined. More than 50% were cytoplasmic and 70% belonged to families of proteins with characteristic binding properties. Proteins were assigned to groups by biological function including regulation (40%), metabolism (26%), inflammation (19%) and structure (15%). More changes were detected at one than seven days after injury at both ages. Seven identified proteins: 14-3-3 epsilon, 14-3-3 gamma, cofilin, alpha enolase, heart fatty acid binding protein (FABP3), brain fatty acid binding protein (FABP7) and ubiquitin demonstrated age-related differential expression and were analysed by qRT-PCR. Changes in mRNA levels for FABP3 at P7+1day and ubiquitin at P28+1day were statistically significant. Immunocytochemical staining showed differences in ubiquitin localization in younger compared to older cords and an increase in oligodendrocyte and neuroglia immunostaining following injury at P28. Western blot analysis supported proteomic results for ubiquitin and 14-3-3 proteins. Data obtained at the two ages demonstrated changes in response to injury, compared to controls, that were different for different functional protein classes. Some may provide targets for novel drug or gene therapies.

ENO1 (Enolase 1, (alpha))

Review on ENO1 (Enolase 1, (alpha)), with data on DNA, on the protein encoded, and where the gene is implicated.

Comparative gene expression profiling analysis of lymphoblastoid cells reveals neuron-specific enolase gene (ENO2) as a susceptibility gene of heroin dependence

Heroin dependence is a complex mental disorder resulting from interactions between genetic and environmental factors. Identifying the susceptibility genes of heroin dependence is the basis for understanding the pathogenesis of heroin dependence. Using a total gene expression microarray, we detected 924 differentially expressed gene transcripts in lymphoblastoid cell lines (LCLs) between 19 male heroin-dependent individuals and 20 male control subjects, including 279 upregulated and 645 downregulated gene transcripts in heroin-dependent individuals. We verified the reduced expression of the neuron-specific enolase gene (ENO2) in heroin-dependent individuals using real-time quantitative polymerase chain reaction and Western blot analysis. We further compared the allele and genotype frequencies of three single nucleotide polymorphisms (SNPs, rs11064464, rs3213433 and rs10849541) of the ENO2 gene between 532 male heroin-dependent individuals and 369 male controls. No significant differences in the allele or genotype frequencies of these three SNPs were detected between these two groups. Nevertheless, we identified a haplotype (T-C-G) derived from these three SNPs significantly underrepresented in heroin-dependent individuals compared with the control group (72.7% versus 75.9%, P<0.032), while two other rare haplotypes (C-A-G and T-C-A) significantly overrepresented in heroin-dependent individuals compared with the control group (P<0.001). Further study, however, did not detect significant differences of the plasma concentration of neuron-specific enolase between these two groups. Our data suggest that the ENO2 gene might be associated with heroin dependence, and reduced ENO2 gene expression may confer increased risk to heroin dependence.

Comparative proteomic analysis of renal tubular epithelial cell injury caused by oxalic acid and calcium oxalate monohydrate

Objective To analyze and identify the differentially expressed proteins in human renal tubular epithelial cells (HK-2) after injury caused by oxalic acid and calcium oxalate monohydrate (COM) crystal, and to explore the potential role of renal tubular cell injury in kidney stone formation.Methods Normal HK-2 cells were cultured in vitro and the culture medium was changed with serum-free medium after cell growth to confluence. Oxalic acid and COM crystals (final concentration at 2 mmol/L and 200 mg/L, respectively) were added in the experimental group. Cells in both groups were then incubated at 37 ℃ for 12 h. The extracted proteins from both groups were separated by two dimensional electrophoresis followed by analysis, and the differentially expressed proteins were identified by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Two identified proteins were then verified by western blot. Results Reproducible two dimensional gel images of the proteins from both groups were successfully obtained. By using LC-ESI-MS/MS, 12 proteins: FK506-binding protein 4, isoform alpha-enolase of alpha-enolase, isoform M1 of pyruvate kinase isozymes M1/M2, ATP synthase subunit alpha, isoform 1 of 3′(2′), 5′-bisphosphate nucleotidase 1, isoform 2 of nucleophosmin, L-lactate dehydrogenase B chain, Budding uninhibited by benzimidazoles 3, Cofilin-1, Fascin, pyIsoform 1 of cytosol aminopeptidase, were identified. The deferentially expressed proteins were related to cellular processes including energy metabolism, cell multiplication, apoptosis, Ca2+ channel activity regulation, cell movement and signal transduction. Western blot verified that higher ENO1 but lower Cofilin-1 expressed in HK-2 cells after the injury. Conclusions High level oxalic acid and COM crystals can cause protein expression profile changes in normal human HK-2 cells. The changes of protein expression may not only protect HK-2 cells from being injured, but also be related to kidney stone formation. Key words: Renal tubular epithelial cell; Oxalate; Calcium oxalate monohydrate; Renal calculus; Proteomics

Continuous neurogenesis in the adult forebrain is required for innate olfactory responses

Although the functional significance of adult neurogenesis in hippocampal-dependent learning and memory has been well documented, the role of such neurogenesis in olfactory activity is rather obscure. To understand the significance of adult neurogenesis in olfactory functions, we genetically ablated newly born neurons by using tamoxifen-treated Nestin-CreER(T2);neuron-specific enolase-diphtheria toxin fragment A (NSE-DTA) mice. In these mice, tamoxifen-inducible Cre recombinase allows the NSE (Eno2) gene to drive DTA expression in differentiating neurons, leading to the efficient ablation of newly born neurons in the forebrain. These mutant mice were capable of discriminating odors as competently as control mice. Strikingly, although control and mutant mice frequently showed freezing behaviors to a fox scent, a predator odor, mutant mice approached this odor when they were conditioned to associate the odor with a reward, whereas control mice did not approach the odor. Furthermore, although mutant males and females showed normal social recognition behaviors to other mice of a different sex, mutant males displayed deficits in male-male aggression and male sexual behaviors toward females, whereas mutant females displayed deficits in fertility and nurturing, indicating that sex-specific activities, which are known to depend on olfaction, are impaired. These results suggest that continuous neurogenesis is required for predator avoidance and sex-specific responses that are olfaction dependent and innately programmed.

Differentiation of vitreous proteome in human eye with proliferative Vitreoretinopathy

Background Proliferative vitreoretinopathy (PVR) is a a common cause of anatomic failure in retinal detachment surgery.Proteomics is the critical method to investigate the different proteins. Objective This study was to search for related vitreous proteins in rhegmatogenous retinal detachment patients with proliferative vitreoretinopathy (PVR)and screen for the related protein expression in the vitreous with PVR. Methods Vitreous samples were obtained from 8 moderate PVR (grade B)eyes and 8 severe PVR(grade C or D)eyes during pars plana vitrectomy (PPV)for the proteomics analysis by two-dimensional gel electrophoresis coupled with mass spectrometry.Normal vitreous from 8 healthy donor eyes served as control.First dimension isoelectric focusing (IEF) was performed with the Pharmacia IPGphor in solvent B using 18 cm non-linear pH 3-10 immobilized pH gradient (IPG)strips.IPG strips were rehydrated with sample,and then IEF was performed.The second dimension 12%sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) was performed after the IPG strips were equilibrated.After silver staining,the gels were analyzed by the 2-DE gel analysis software.The matched spots were excised and tryptica digested in-gel.The peptides were analyzed by MALDI-TOF-MS and the MS/MS spectral were searched against the human protein databases using MASCOT.This study process complied with the Declaration of Helsinki.Wfitten informed consent was obtained from each patient prior to this trial. Results In the current study,a total of 47,184 and 336 protein spots were detected from the normal donor eyes,moderate PVR eyes and severe PVR eyes,respectively,by 2-DE gels.Among 13 protein spots with significant differences in the 3 groups,7 types of proteins were successfully identified.The results indicated that Enolase 2 was a specific protein for normal donor vitreous.whereas transthyretin monomer and retinol-binding protein(RBP) chain B were distinct proteins found in severe PVR vitreous.and prostaglandin D synthase and RBP3 precursor were common proteins with up-regulated expression in vitreous samples with moderate PVR,but down-regulated levels in vitreous samples with severe PVR.The levels of albumin and transferrin in vitreous showed dynamic elevation with the enhancement of severity of PVR.Conclusion The changes in the vitreous proteome imply that some types of vitreous proteins degrade and some common serum proteins are expressed in the vitreous body with PVR,which indicated the disruption of the blood retinal barrier in the pathogenesis of PVR. Key words: Proteome; Proliferative vitreoretinopathy; Vitreous

A potential role for co‐amplification of other oncogenes with EGFR in the control of metabolism in glioblastomas

Based on EGFR's frequent amplification in glioblastomas, gains in copy numbers for it and other oncogenes were investigated for associations with expression of genes that encode mediators of glycolysis. ATP citrate lyase (ACLY) was included due to its protein's cleavage of cytosolic citric acid, a glycolysis inhibitor. RNA and DNA were isolated from 22 glioblastomas (adults) and analyzed with real‐time quantitative PCR (RT‐qPCR) and multiplex ligation‐dependent probe amplification (MLPA). Transcript levels of ACLY, enolase 1 (ENO1), and other genes were analyzed with delta delta crossing threshold methodology. RT‐qPCR results were normalized with actin B and glyceraldehyde phosphate dehydrogenase using normal brain RNA. Expression of ACLY correlated well with ENO1. MLPA data for 78 oncogenes were normalized against normal DNA. At least 15 oncogenes showed ≥3‐fold amplifications in 14 of 22 glioblastomas. FISH confirmed results for EGFR in 18 cases tested. EGFR was amplified alone and combined with other oncogenes in 7 &amp; 4 tumors, respectively. A trend (p = 0.1167, Fisher exact test) was detected for increased ACLY expression when EGFR was co‐amplified with CDK6, CDK4, MOS, CYP27B1 &amp;/or PDGFRA, but not alone. Co‐amplification of other oncogenes with EGFR may correlate with regulation of glycolysis.

Bioinformatic studies of vertebrate enolases: multifunctional genes and proteins

Bioinformatic studies of vertebrate enolases: multifunctional genes and proteins Roger S HolmesSchool of Biomolecular and Physical Sciences, Griffith University, Nathan, QLD, AustraliaAbstract: Enolase (ENO) genes and proteins (ENO; EC 4.2.1.11) serve multiple functions in the body, including catalyzing 2-phospho-d-glycerate hydro-lyase activity in glycolysis, assisting hypoxia tolerance, tumor suppression, plasminogen and DNA binding, and acting as a lens crystallin. Comparative ENO amino acid sequences and structures and ENO gene locations were examined using data from several vertebrate genome projects. Vertebrate ENO1, ENO2, and ENO3 genes usually contained 11 coding exons, while ENO4 (encoding an ENO-like protein, ENOLL) usually contained 14 coding exons. Vertebrate ENOF1 (or ENO5) genes encode an antisense RNA, which may regulate mitochondrial thymidylate synthase activity that contained 12&ndash;15 coding exons. Vertebrate ENO1, ENO2, and ENO3 sequences shared 78%&ndash;98% identities but only 19%&ndash;24% with ENO4 and &gt;10% predicted sequence identities with vertebrate ENOF1. Sequence alignments, key amino acid residues, and conserved predicted secondary and tertiary structures were examined, including active site residues (absent in ENO4 and ENOF1) and sites for Mg2+ and plasminogen binding and for acetylation and phosphorylation. The predicted ENO4 structure contained three N-terminal a-helices, two &szlig;-sheets, a poly-proline segment, and an extended C-terminal sequence in addition to the typical a/&szlig; barrel structure reported for ENO1&ndash;3 sequences. Potential transcription factor binding sites (TFBS) and CpG islands for regulating ENO gene expression were identified. Human ENO1, ENO2, ENO3, and ENOF1 genes each contained CpG islands in the gene promoter regions consistent with higher-than-average levels of expression. Human ENO3 and ENO1 gene promoters also contained a diverse range of TFBS. The ENO4 gene promoter comprised a CpG island and several TFBS, including AHR1 in the 5&#39;-UTR region, which may suggest a role for ENO4 in aryl hydrocarbon ligand binding or metabolism. Phylogeny studies of vertebrate ENO1, ENO2, and ENO3 genes and enzymes suggested that they originated in a vertebrate ancestor from gene duplication events of an ancestral ENO1-like gene &gt;500 million years ago.Keywords: vertebrate, amino acid sequence, enolase, evolution, bioinformatics

Differential Proteomic Analysis of Renal Cell Carcinoma Tissue Interstitial Fluid

Renal cell carcinoma (RCC), the most common type of kidney cancer, currently has no biomarker of clinical utility. The present study utilized a mass spectrometry-based proteomics workflow for identifying differentially abundant proteins in RCC by harvesting shed and secreted proteins from the tumor microenvironment through sampling tissue interstitial fluid (TIF) from radical nephrectomies. Matched tumor and adjacent normal kidney (ANK) tissues were collected from 10 patients diagnosed with clear cell RCC. One-hundred thirty-eight proteins were identified with statistically significant differential abundances derived by spectral counting in tumor TIF when compared to ANK TIF. Among those proteins with elevated abundance in tumor TIF, nicotinamide n-methyltransferase (NNMT) and enolase 2 (ENO2) were verified by Western blot and selected reaction monitoring (SRM). The presence of ENO2 and thrombospondin-1 (TSP1) were verified as present and at elevated abundance in RCC patient serum samples as compared to a pooled standard control by enzyme-linked immunosorbent assay (ELISA), recapitulating the relative abundance increase in RCC as compared with ANK TIF.

Gonadotropins up-regulate the expression of enolase 2, but not enolase 1, in the rat ovary

It has been demonstrated that the glycolytic enzymes, enolase 1 (ENO1) and enolase 2 (ENO2), are expressed in the rat ovary. In the present study, we found that mRNA levels of ovarian ENO2 but not ENO1 in normal cycling adult female rats changed significantly during the estrous cycle: ovarian ENO2 mRNA levels at metestrus were lower than those at estrus. Single injection of human CG (hCG) or equine CG (eCG) into immature (3 week old) rats up-regulated ovarian expression of ENO2. hCG mainly increased ENO2 expression in oocytes and theca cells of preantral and antral follicles, and eCG did in theca cells of these follicles. In contrast, hCG and eCG did not affect the expression of ENO1, which was mainly expressed in granulosa cells. These results suggest that endogenous gonadotropins up-regulate expression of ENO2 in oocytes and theca cells of preantral and antral follicles, which would activate glycolysis in these cells. It is also suggested that the activated glycolysis is necessary for ovarian functions such as follicle growth and maturation, and hormone production.

Identification of CM1 as a Pathogenic Factor in Inflammatory Diseases and Cancer

BackgroundCM1 (centrocyte/-blast marker 1) was defined by a mAb against concanavalin A (Con A) activated PBMC. It is expressed in germinal center of human tonsil and on the surface of activated PBMC as well as cancer cells. Recently, increased productions of pro-inflammatory mediators were detected from activated PBMC by CM1 ligation.MethodsHowever, there is a limitation to explain the exact role of CM1 on inflammation and its related mechanisms, since the identity of CM1 is still not clarified. In our previous study, we have already confirmed that soluble form of CM1 was produced by Raji. Therefore, we performed Q-TOF analysis after immunoprecipitation of concentrated Raji culture supernatant using anti-CM1 mAbs.ResultsAs a result, we found that CM1 is identical to enolase-1(ENO1), a glycolytic enzyme, and we confirmed that results by silencing ENO1 using siRNA. It was also confirmed through competition assay between anti-CM1 and anti-ENO1 mAbs. Finally, we investigated the possible role of CM1 in inflammatory response and cancer. The ligation of CM1 on Raji cells with anti-CM1 mAbs induces the extensive production of prostaglandin E2(PGE2). In addition, the increased activity of matrix metalloproteinase (MMP)-2/9 was shown in NCI-N87, stomach cancer cell line by CM1 stimulation.ConclusionCM1 is identical to ENO1 and it might be an important role in the regulation of inflammatory responses.

Effects of Salvianolic Acid B on Protein Expression in Human Umbilical Vein Endothelial Cells

Salvianolic acid B (Sal B), a pure water-soluble compound extracted from Radix Salviae miltiorrhizae, has been reported to possess potential cardioprotective efficacy. To identify proteins or pathways by which Sal B might exert its protective activities on the cardiovascular system, two-dimensional gel electrophoresis-based comparative proteomics was performed, and proteins altered in their expression level after Sal B treatment were identified by MALDI-TOF MS/MS. Human umbilical vein endothelial cells (HUVECs) were incubated at Sal B concentrations that can be reached in human plasma by pharmacological intervention. Results indicated that caldesmon, an actin-stabilizing protein, was downregulated in Sal B-exposed HUVECs. Proteins that showed increased expression levels upon Sal B treatment were vimentin, T-complex protein 1, protein disulfide isomerase, tropomyosin alpha, heat shock protein beta-1, UBX domain-containing protein 1, alpha enolase, and peroxiredoxin-2. Additionally, Sal B leads to increased phosphorylation of nucleophosmin in a dose-dependent manner and promotes proliferation of HUVECs. We found that Sal B exhibited a coordinated regulation of enzymes and proteins involved in cytoskeletal reorganization, oxidative stress, and cell growth. Our investigation would provide understanding to the endothelium protection information of Sal B.

Identification biomarkers for cervical cancer in peripheral blood lymphocytes by oligonucleotide microarrays

To identify the molecular biomarkers for cervical cancer in peripheral blood lymphocytes by oligonucleotide microarrays. Human genome oligonucleotide microarray analysis included 4 early-stage cervical cancer patients and 3 controls. The selected genes from the microarray analysis were validated in additional 20 early-stage cervical cancer patients and 15 controls by real-time reverse-transcription polymerase chain reaction (RT-PCR). Genes identified by gene selection program were expressed differently in the blood samples of early-stage cervical cancer from those of healthy controls. To validate the gene expression data, 5 genes were analyzed by real-time RT-PCR. In three of 5 identified genes, tenascin-c, nucleolin, and enolase 2 (ENO2) showed a significant up-regulation in blood samples of early-stage cervical cancer patients versus that of the controls. The up-regulation of tenascin-c, nucleolin and ENO2 in peripheral blood may be used to identify novel blood biomarkers for detecting cervical cancer in a clinically accessible surrogate tissue. Thus it may offer a possibility of developing a non-invasive and predictive diagnostic tool for the disease.