MicroRNA-99a inhibits hepatocellular carcinoma growth and correlates with prognosis of patients with hepatocellular carcinoma

In our in-depth analysis carried out by the Illumina Solexa massive parallel signature sequencing, microRNA-99a (miR-99a) was found to be the sixth abundant microRNA in the miRNome of normal human liver but was markedly down-regulated in hepatocellular carcinoma (HCC). Compelling evidence has suggested the important roles of microRNAs in HCC development. However, the biological function of miR-99a deregulation in HCC remains unknown. In this study, we found that miR-99a was remarkably decreased in HCC tissues and cell lines. Importantly, lower miR-99a expression in HCC tissues significantly correlated with shorter survival of HCC patients, and miR-99a was identified to be an independent predictor for the prognosis of HCC patients. Furthermore, restoration of miR-99a dramatically suppressed HCC cell growth in vitro by inducing the G(1) phase cell cycle arrest. Intratumoral injection of cholesterol-conjugated miR-99a mimics significantly inhibited tumor growth and reduced the α-fetoprotein level in HCC-bearing nude mice. Insulin-like growth factor 1 receptor (IGF-1R) and mammalian target of rapamycin (mTOR) were further characterized as the direct targets of miR-99a. Furthermore, protein levels of IGF-1R and mTOR were found to be inversely correlated with miR-99a expression in HCC tissues. miR-99a mimics inhibited IGF-1R and mTOR pathways and subsequently suppressed expression of cell cycle-related proteins, including cyclin D1 in HCC cells. Conclusively, miR-99a expression was frequently down-regulated in HCC tissues and correlates with the prognosis of HCC patients, thus proposing miR-99a as a prospective prognosis predictor of HCC. miR-99a suppresses HCC growth by inducing cell cycle arrest, suggesting miR-99a as potential tumor suppressor for HCC therapeutics.     

Structure, localization and function of FanF, a minor component of K99 fibrillae of enterotoxigenic Escherichia coii

The DNA sequence of the K99 fanF gene, encoding FanF, was determined. An open reading frame of 999 bp was found. The primary structure of FanF was deduced and analysis revealed the presence of a signal sequence of 22 amino acid residues. The mature protein contains 311 amino acid residues (Mr 33,905 D). The amino acid sequence of FanF showed similarity with the K88ab major subunit FaeG. A specific mouse antiserum against FanF was prepared by constructing and purifying a hybrid Cro-LacZ-FanF protein. Minicell analysis, immunoblotting and immunoelectronmicroscopy revealed a pool of FanF in the periplasm of K99-producing cells and showed, furthermore, that FanF is a minor component of K99 fibrillae, present at the top and in or along the shaft of the K99 fibrillar structures. A fanF mutant plasmid was constructed. Cells harbouring this plasmid produced all K99-specific proteins, except FanF, but produced 0.1% of the K99 fibrillae relative to 'normal' K99-producing cells. Electron microscopic observations showed that cells defective in fanF produce only a few (apparently short) K99 fibrillae. FanF, therefore, was supposed to play a role in initiation and elongation of K99 fibrillae formation. Thin-layer chromatography experiments involving purified receptor material showed that FanF is not required for binding of K99 fibrillae to the ganglioside receptor. Fibrillae produced by an adhesion-negative strain carrying a mutation in the K99 major fibrillar subunit were shown to contain a normal amount of FanF.     

Heterologous expression, purification, and characterization of an α-mannosidase belonging to glycoside hydrolase family 99 of Shewanella amazonensis

Shewanella amazonensis α-mannosidase (Sama99), a member of glycoside hydrolase family 99, was expressed in Escherichia coli. The purified Sama99 hydrolyzed pyridylamino (PA)-sugars, Glc?Man?GlcNAc?-PA, and Glc?Man?GlcNAc?-PA, and the product was probably a pyridylamino-decasaccharide in both cases. The mode of action of Sama99 was found to be essentially identical to that of rat endo-α-1,2-mannosidase, but the specificity of Sama99 was low.     

Pulmonary clearance of inhaled 99mTc-DTPA: effects of surfactant depletion by lung lavage

The influence of surfactant depletion on clearance from the lungs of inhaled technetium-99m-labeled diethylenetriamine pentaacetate (99mTc-DTPA) was studied in rabbits. Surfactant was removed by repeated lung lavage with isotone saline. To minimize structural damage to the lungs, pressure generated insufflation with short expiration was utilized. Aerosolized 99mTc-DTPA was administered via a bag-in-bottle system. Radioactivity was measured with a gamma camera and time-activity curves were obtained over the base of the right lung. Six nonlavaged rabbits served as controls. In six lavaged rabbits clearance of 99mTc-DTPA was significantly faster than in controls. In three rabbits given natural surfactant into the trachea after lung lavage, 99mTc-DTPA was eliminated faster than in controls but slower than in surfactant-depleted animals. The results indicate a role of surfactant on clearance rate of 99mTc-DTPA from rabbit lungs. Measurements of 99mTc-DTPA clearance may be useful in studying the function of the surfactant system in different lung disorders.     

CD99 is a key mediator of the transendothelial migration of neutrophils

Transendothelial migration of leukocytes is a critical event for inflammation, but the molecular regulation of this event is only beginning to be understood. PECAM (CD31) is a major mediator of monocyte and neutrophil transmigration, and CD99 was recently defined as a second mediator of the transmigration of monocytes. Expression of CD99 on the surface of circulating polymorphonuclear cells (PMN) is low compared with expression of CD99 on monocytes or expression of PECAM on PMN. We demonstrate here that, despite low expression of CD99, Fab of Abs against CD99 blocked over 80% of human neutrophils from transmigrating across HUVEC monolayers in an in vitro model of inflammation. Blocking CD99 on either the neutrophil or endothelial cell side resulted in a quantitatively equivalent block, suggesting a homophilic interaction between CD99 on the neutrophil and CD99 on the endothelial cell. Blocking CD99 and PECAM together resulted in additive effects, suggesting the two molecules work at distinct steps. Confocal microscopy confirmed that CD99-blocked neutrophils lodged in endothelial cell junctions at locations distal to PECAM-blocked neutrophils. The CD99-blocked PMN exhibited dynamic lateral movement within endothelial cell junctions, indicating that only the diapedesis step was blocked by interference with CD99. Anti-CD99 mAb also blocked PMN transmigration in a second in vitro model that incorporated shear stress. Taken together, the evidence demonstrates that PECAM and CD99 regulate distinct, sequential steps in the transendothelial migration of neutrophils during inflammation.     

Synthesis and biodistribution of a novel (99m)TcN complex of ciprofloxacin dithiocarbamate as a potential agent for infection imaging

The ciprofloxacin dithiocarbamate (CPFXDTC) was synthesized and radiolabeled with [(99m)TcN](2+) intermediate to form the (99m)TcN-CPFXDTC complex in high yield (>95%). No decomposition of the complex at room temperature was observed over a period of 6 h. Its partition coefficient indicated that it was a good lipophilic complex. The bacterial binding assay studies showed (99m)TcN-CPFXDTC had a better binding affinity as compared with (99m)Tc-ciprofloxacin. Biodistribution results in induced infection mice showed (99m)TcN-CPFXDTC had higher uptake at the sites of infection and better abscess/blood ratio than that of (99m)Tc-ciprofloxacin, suggesting (99m)TcN-CPFXDTC would be a novel potential infection imaging agent.     

Construction and characterization of phage-displayed leukocyte surface molecule CD99

The phage display technique has been described for the production of various recombinant molecules. In the present report, we used this technique to display a leukocyte surface molecule, CD99. PCR subcloning of CD99 cDNA from the mammalian expression vector pCDM8 to the phagemid expression vector pComb3HSS was performed. The resulting phagemid, pComb3H-CD99, was transformed into Escherichia coli XL-1 Blue. CD99 was displayed on the phage particles following infection of the transformed E. coli with the filamentous phage VCSM13. Using sandwich ELISA, the filamentous phage-displayed CD99 was captured by a CD99 monoclonal antibody (mAb) then detected with anti-M13 conjugated to horseradish peroxidase, confirming that the CD99 molecule was displayed on the phage particles. The CD99-phages inhibited induction of Jurkat cell aggregation by CD99 mAb MT99/1. Proper folding of the displayed CD99 bioactive domain was inferred from this finding. Our results demonstrate that the phage display technique can be applied to the generation of full-length CD99 molecules. The phage carrying this cell surface protein will be useful for identification of its counter receptor or ligand.     

Technetium labeling of monoclonal antibodies with functionalized BATOs. 1. TcCl(DMG)3PITC

BATO (boronic acid adduct of technetium dioximes) complexes, TcCl(dioxime)3BR, were prepared in which the boron substituent (R) was the protein-reactive m-phenyl isothiocyanate (PITC). The 99TcCl(dioxime)3PITC complexes [dioxime = dimethylglyoxime (DMG) or cyclohexanedione dioxime (CDO)] were prepared from 99Tc(dioxime)3(mu-OH)SnCl3 and characterized. The X-ray crystal structure of 99TcCl(DMG)3PITC was determined. The 99mTc complexes were prepared from 99mTcO4- in a process using a freeze-dried kit, either in a one-step procedure or via 99mTcCl(dioxime)3. Initial labeling studies with 99mTcCl(dioxime)3PITC were performed on glycine and polylysine and, subsequently, on mouse IgG and the B72.3 monoclonal antibody. Covalent attachment of 99mTcCl(DMG)3PITC to B72.3 was demonstrated by SDS-PAGE electrophoresis. B72.3 labeled with 99mTcCl(DMG)3PITC displayed high binding to a TAG 72 affinity column and had a distribution in normal mice similar to that reported for iodine-labeled B72.3.     

8-cyclopentadienyltricarbonyl 99mtc 8-oxooctanoic acid: a novel radiotracer for evaluation of medium chain fatty acid metabolism in the liver

8-Cyclopentadienyltricarbonyl 99mTc 8-oxooctanoic acid (99mTc-CpTTOA; 1a) was synthesized for evaluation of medium chain fatty acid metabolism in the liver. 99mTc-CpTTOA was prepared in high radiochemical yield (50-63%) by a double ligand transfer reaction of methyl 8-ferrocenyl-8-oxooctanoate and Na99mTcO4 in the presence of CrCl3 and Cr(CO)6, followed by hydrolysis. This radiotracer was shown to be stable (>90% at 6 h) when incubated with human serum. Aqueous extraction of the radioactivity from the liver and blood samples of mice suggested that 99mTc-CpTTOA was mainly metabolized via beta-oxidation in the liver, and the radioactivity was retained longer in CCl4-treated mice than in control mice, possibly due to impaired beta-oxidation in the former. Planar images of rats injected with 99mTc-CpTTOA showed accumulation of the radioactivity in the liver, kidneys, and bladder with rapid hepatic clearance as a function of time. Analysis of the metabolites from the liver and urine samples of rats further supported that 99mTc-CpTTOA was metabolized to 4-cyclopentadienyltricarbonyl 99mTc 4-oxobutanoic acid (99mTc-CpTTBA; 1c) via beta-oxidation. The results suggested that this radiotracer might be of valuable use in the evaluation of fatty acid metabolism in the liver.     

The activation of CD99 inhibits cell-extracellular matrix adhesion by suppressing β1 integrin affinity

CD99 is known to be involved in the regulation of cell-cell adhesion. However, it remains unclear whether CD99 controls cell-extracellular matrix adhesion. In this study, the effects of CD99 activation on cell-extracellular matrix adhesion were investigated. It was found that engagement of CD99 with the stimulating antibody YG32 downregulated the adhesion of MCF-7 cells to fibronectin, laminin and collagen IV in a dose-dependent manner. The CD99 effect on cell-ECM adhesion was inhibited by overexpression of the dominant negative form of CD99 or CD99 siRNA transfection. Treatment of cells with Mn(2+) or by β(1) integrin-stimulating antibody restored the inhibitory effect of CD99 on cell-ECM adhesion. Cross-linking CD99 inactivated β(1) integrin through conformational change. CD99 activation caused dephosphorylation at Tyr-397 in FAK, which was restored by the β(1) stimulating antibody. Taken together, these results provide the first evidence that CD99 inhibits cell-extracellular matrix adhesion by suppressing β(1) integrin affinity. [BMB reports 2012; 45(3): 159-164].     

Control of temperature-dependent synthesis of K99 fimbriae

The influence of temperature on the production of K99 fimbriae by Escherichia coli was determined in cultures growing at constant specific growth rate in continuous cultures. In a wild type strain, in which the K99 operon is present on a low copy number plasmid, low cultivation temperature repressed the K99 production. This temperature-dependent production was not observed after introduction of multicopies of the regulatory region of the K99 operon into this strain, nor in E. coli K12 harbouring a recombinant, multicopy plasmid encoding the K99 operon. These results are in agreement with a regulation model in which a regulatory factor, most likely a repressor, inhibits expression of the K99 operon at low temperatures.     

Engagement of CD99 triggers the exocytic transport of ganglioside GM1 and the reorganization of actin cytoskeleton

We studied the role of lipid rafts and actin cytoskeleton in CD99-mediated signaling to elucidate the mechanism of protein transport upon CD99 engagement. CD99 engagement in Jurkat cells elicited the exocytic transport of GM1 as well as several surface molecules closely related with CD99 functions. In addition, CD99 molecules were rapidly incorporated into lipid rafts and appeared to rearrange the actin cytoskeleton upon CD99 stimulation. Association of CD99 with actin cytoskeleton was inhibited by methyl-β-cyclodextrin, while CD99-mediated GM1 clustering was inhibited by cytochalasin D. Therefore, we suggest that CD99 may play a role in the vesicular transport of transmembrane proteins and lipid rafts from the intracellular location to the cell surface, possibly by effecting actin cytoskeleton reorganization.     

Validation of ⁹⁹mTechnetium-labeled mebrofenin hepatic extraction method to quantify meal-induced splanchnic blood flow responses using a porcine model

The aim of this study was to evaluate the measurement of the total splanchnic blood flow (SBF) using a clinical diagnostic method based on Fick's principle and hepatic extraction of 99mTc-mebrofenin (99mTc-MBF) compared with a paraaminohippuric acid (pAH) dilution method in a porcine model. Another aim was to investigate whether enterohepatic cycling of 99mTc-MBF affected the SBF measurement. Five indwelling catheters were placed in each pig (n = 15) in the portal, mesenteric, and hepatic veins, as well as in the aorta and the vena cava. The SBF was measured using both methods. The portal blood flow; the intestinal and hepatic oxygen uptake; the net fluxes of oxygen, lactate, and glucose; and the extraction fraction (EF) of 99mTc-MBF were measured before and for 70 min after feeding. The mean baseline SBF was 2,961 ml/min vs. 2,762 ml/min measured by pAH and 99mTc-MBF, respectively, and increased significantly to 3,977 ml/min and 3,981 ml/min postprandially. The hepatic EF of 99mTc-MBF decreased from 40% at the start of the investigation to 16% 70 min after feeding. The arterial-portal difference in 99mTc-MBF concentration was 0.21% (P = 0.48), indicating no intestinal extraction or metabolism. The clinical method for measuring the SBF based on hepatic 99mTc-MBF extraction is robust compared with the indicator dilution method, despite the decrease seen in hepatic extraction of 99mTc-MBF. Because there was no difference in the content of 99mTc-MBF between the arterial and portal vein plasma, the SBF can be calculated from an arterial and a hepatic vein sample.     

Targeting arterial wall sulfated glycosaminoglycans in rabbit atherosclerosis with a mouse/human chimeric antibody

The progression of atherosclerosis is favored by increasing amounts of chondroitin sulfate proteoglycans in the artery wall. We previously reported the reactivity of chP3R99 monoclonal antibody (mAb) with sulfated glycosaminoglycans and its association with the anti-atherogenic properties displayed. Now, we evaluated the accumulation of this mAb in atherosclerotic lesions and its potential use as a probe for specific in vivo detection of the disease. Atherosclerosis was induced in NZW rabbits (n = 14) by the administration of Lipofundin 20% using PBS-receiving animals as control (n = 8). Accumulation of chP3R99 mAb in atherosclerotic lesions was assessed either by immunofluorescence detection of human IgG in fresh-frozen sections of aorta, or by immunoscintigraphy followed by biodistribution of the radiotracer upon administration of ^99mTc-chP3R99 mAb. Immunofluorescence studies revealed the presence of chP3R99 mAb in atherosclerotic lesions 24 h after intravenous administration, whereas planar images showed an evident accumulation of ^99mTc-chP3R99 mAb in atherosclerotic rabbit carotids. Accordingly, ^99mTc-chP3R99 mAb uptake by lesioned aortic arch and thoracic segment was increased 5.6-fold over controls and it was 3.9-folds higher in carotids, in agreement with immunoscintigrams. Moreover, the deposition of ^99mTc-chP3R99 mAb in the artery wall was associated both with the presence and size of the lesions in the different portions of evaluated arteries and was greater than in non-targeted organs. In conclusion, chP3R99 mAb preferentially accumulates in arterial atherosclerotic lesions supporting the potential use of this anti-glycosaminoglycans antibody for diagnosis and treatment of atherosclerosis.     

Comparison of three tracers for detecting lung epithelial injury in anesthetized sheep

We compared the ability of three aerosolized tracers to discriminate among control, lung inflation with a positive end expired pressure of 10 cmH2O, lung vascular hypertension and edema without lung injury, and lung edema with lung injury due to intravenous oleic acid. The tracers were 99mTc-diethylenetriaminepentaacetate (99mTc-DTPA, mol wt 492), 99mTc-human serum albumin (99mTc-ALB, mol wt 69,000), and 99mTc-aggregated albumin (99mTc-AGG ALB, mol wt 383,000). 99mTc-DTPA clearance measurements were not able to discriminate lung injury from lung inflation. The 99mTc-AGG ALB clearance rate was unchanged by lung inflation and increased slightly with lung injury. The 99mTc-ALB clearance rate (0.06 +/- 0.02%/min) was unchanged by lung inflation (0.09 +/- 0.02%/min, P greater than 0.05) or 4 h of hypertension without injury (0.09 +/- 0.04%/min, P greater than 0.05). Deposition of 99mTc-ALB within 15 min of the administration of the oleic acid increased the clearance rate to 0.19 +/- 0.06%/min, which correlated well with the postmortem lung water volume (r = 0.92, P less than 0.01). This did not occur when there was a 60-min delay in the deposition of 99mTc-ALB. We conclude that 99mTc-ALB is the best indicator for studying the effects of lung epithelial injury on protein and fluid transport into and out of the air spaces of the lungs in a minimally invasive manner.     

Technetium-99m-labeled medium-chain fatty acid analogues metabolized by beta-oxidation: radiopharmaceutical for assessing liver function.

External imaging of energy production activity of living cells with 99mTc-labeled compounds is a challenging task requiring good design of 99mTc-radiopharmaceuticals. On the basis of our recent findings that 11C- and 123I-labeled medium-chain fatty acids are useful for measuring beta-oxidation activity of hepatocytes, we focused on development of 99mTc-labeled medium-chain fatty acid analogues that reflect beta-oxidation activity of the liver. In the present study, monoamine-monoamide dithiol (MAMA) ligand and triamido thiol (MAG) ligand were chosen as chelating groups because of the stability and size of their complexes with 99mTc and their ease of synthesis. Each ligand was attached to the omega-position of hexanoic acid (MAMA-HA and MAG-HA, respectively). In biodistribution studies, [99mTc]MAMA-HA showed high initial accumulation in the liver followed by clearance of the radioactivity in the urine. Analysis of the urine revealed [99mTc]MAMA-BA as the sole radiometabolite. Furthermore, when [99mTc]MAMA-HA was incubated with living liver slices, generation of [99mTc]MAMA-BA was observed. However, [99mTc]MAMA-HA remained intact when the compound was incubated with liver slices in the presence of 2-bromooctanoate, an inhibitor of beta-oxidation. The findings in this study indicated that [99mTc]MAMA-HA was metabolized by beta-oxidation after incorporation into the liver. On the other hand, poor hepatic accumulation was observed after administration of [99mTc]MAG-HA.     

Amino acid substitution at position 99 affects the rate of CRP subunit exchange

We investigated the characteristics of CRP having amino acid substitutions at position 99. Analysis of amino acid residue proximity to cAMP in molecular dynamics (MD) simulations of the CRP:(cAMP)(2) complex [García, A. E., and Harman, J. G. (1996) Protein Sci. 5, 62-71] showed repositioning of tyrosine 99 (Y99) to interact with the equatorial exocyclic oxygen atom of cAMP. To test the role of Y99 in cAMP-mediated CRP activation, Y99 was substituted with alanine (A) or phenylalanine (F). Cells that contained the WT or mutant forms of CRP induced beta-galactosidase in the presence of cAMP. Purified WT, Y99A, and Y99F CRP showed only a 3- to 4-fold difference in cAMP affinity. There were no apparent differences between the three forms of CRP in cAMP binding cooperativity, in CRP:(cAMP)(1) complex binding to lacP DNA, in the formation of CRP:cAMP:RNAP complexes at lacP, or in CRP efficacy in mediating lacP activity in vitro. The apo-form of Y99A CRP was more sensitive to protease than the apo-form of either WT CRP or Y99F CRP. Whereas the WT or Y99F CRP:(cAMP)(1) complexes were cleaved by protease at hinge-region peptide bonds, the Y99A CRP:(cAMP)(1) complex was cleaved at peptide bonds located at the subunit interface. The rates of subunit exchange for Y99A CRP, both in the apo-form and in a 1:1 complex with cAMP, were significantly greater than that measured for WT CRP. The results of this study show that tyrosine 99 contributes significant structural stability to the CRP dimer, specifically in stabilizing subunit association.     

Effect of glucose and amino acids on expression of K99 antigen in Escherichia coli

K99 antigen production by enterotoxigenic Escherichia coli strains of bovine origin was investigated by slide agglutination and in vitro attachment to intestinal villi. Work with two strains (B41 and B44) showed that on minimal medium M2, K99 antigen was not repressed by a high concentration of glucose (2%, w/v). Growth on synthetic or complex medium did not affect K99 antigen detection, which was independent of capsular antigens, and its synthesis was not repressed by Casamino acids or glucose. A survey of 12 strains revealed two groups: in one group K99 antigen production was constitutive on basal medium without glucose, and in the second group K99 antigen was produced only in the presence of glucose. Immunoelectrophoresis patterns, and the results of slide agglutination and attachment tests, were dependent upon K99 type, whereas haemagglutination patterns were not.