C-Terminal Farnesylation of UCH-L1 Plays a Role in Transport of Epstein-Barr Virus Primary Oncoprotein LMP1 to Exosomes.

Increasing evidence shows that exosomes are key regulators in cancer cell-to-cell communication. Several reports on Epstein-Barr virus (EBV)-related malignancies demonstrate that latent membrane protein 1 (LMP1) secreted by exosomes derived from EBV- or LMP1-positive cells can promote cancer progression and metastasis. However, the mechanism by which LMP1 is loaded into exosomes is still poorly understood. Here, we examined whether the process of LMP1 loading into exosomes is linked to the multifunctional molecule of the ubiquitin system-ubiquitin C-terminal hydrolase-L1 (UCH-L1). For the first time, we demonstrate that LMP1 is physically associated with UCH-L1 and that directing of LMP1 to exosomes is mediated by C-terminal farnesylation of UCH-L1. Additionally, we found that the FTI-277 farnesyltransferase inhibitor reduces motility- and anchorage-independent growth of EBV-positive cells in functional assays. On the basis of our results, we conclude that C-terminal farnesylation of UCH-L1 is one of the key mechanisms by which LMP1 is sorted to exosomes. We hypothesize that inhibition of farnesylation with specific small-molecule inhibitors blocks exosome-mediated transfer of prometastatic molecules such as LMP1 during cancer cell-to-cell communications and thereby impedes the process of cancer invasion. IMPORTANCE Exosomes are small vesicles that cells secrete into the extracellular space, and there is increasing evidence that they have pivotal roles in cell-to-cell communication in malignancy. It is reported also that EBV-associated malignant cells, including those derived from nasopharyngeal carcinoma (NPC) and B-cell lymphoma, secrete exosomes. These EBV-related exosomes may contain viral products such as latent membrane protein 1 (LMP1) and may contribute to cancer progression. The aim of this study was to investigate the mechanism by which those viral products are loaded in exosomes. In this study, we show for the first time that ubiquitin C-terminal hydrolase-L1 (UCH-L1) and its C-terminal farnesylation, a posttranslational lipid modification, contribute to this mechanism. Our results also suggest that inhibition of UCH-L1 farnesylation is a potential therapeutic target against cancer metastasis and invasion.

Exosomal HIF1α supports invasive potential of nasopharyngeal carcinoma-associated LMP1-positive exosomes.

It has emerged recently that exosomes are potential carriers of pro-tumorigenic factors that participate in oncogenesis. However, whether oncogenic transcription factors are transduced by exosomes is unknown. Hypoxia-inducible factor-1α (HIF1α) transcriptionally regulates numerous key aspects of tumor development and progression by promoting a more aggressive tumor phenotype, characterized by increased proliferation and invasiveness coupled with neoangiogenesis. It has been shown that the principal oncoprotein of Epstein-Barr virus (EBV), latent membrane protein 1 (LMP1), drives oncogenic processes and tumor progression of the highly invasive EBV malignancy, nasopharyngeal carcinoma (NPC). We now demonstrate that endogenous HIF1α is detectable in exosomes and that LMP1 significantly increases levels of HIF1α in exosomes. HIF1 recovered from exosomes retains DNA-binding activity and is transcriptionally active in recipient cells after exosome uptake. We also show that treatment of EBV-negative cells with LMP1-exosomes increases migration and invasiveness of NP cell lines in functional assays, which correlates with the phenotype associated with epithelial-mesenchymal transition (EMT). In addition, we provide evidence that HIF1α itself participates in exosome-mediated pro-metastatic effects in recipient cells, as exosome-mediated delivery of active and inactive forms of HIF1α results in reciprocal changes in the expression of E- and N-cadherins associated with EMT. Further, immunohistochemical analysis of NPC tumor tissues revealed direct correlation between protein levels of LMP1 and of the endosome/exosome marker tetraspanin, CD63, which suggests an increase in exosome formation in this EBV-positive malignancy. We hypothesize that exosome-mediated transfer of functional pro-metastatic factors by LMP1-positive NPC cells to surrounding tumor cells promotes cancer progression.

Descriptive analysis of a novel health care approach: reverse colocation-primary care in a community mental health "home".

OBJECTIVE: Persons with serious mental illness have increased rates of chronic medical conditions, have limited access to primary care, and incur significant health care expenditures. Few studies have explored providing medical care for these patients in the ambulatory mental health setting. This study describes a real-world population of mental health patients receiving primary care services in a community mental health clinic to better understand how limited primary care resources are being utilized. METHOD: Chart review was performed on patients receiving colocated primary care (colocation group, N = 143) and randomly chosen patients receiving mental health care only (mental-health group, N = 156) from January 2006 through June 2011. Demographic and mental and physical health variables were assessed. RESULTS: Compared to the mental-health group, the colocation patients had more psychiatric hospitalizations (mean = 1.07 vs 0.23, P < .01), were more likely to be homeless (P < .01), and were more likely to require intensive case management (P < .01). Interestingly, the colocation group was not more medically ill than the mental-health group on key metabolic measures, including mean body mass index (colocation = 27.8 vs mental-health = 28.7, P = .392), low-density liprotein (colocation = 110.0 vs mental-health = 104.4, P = .480), and glucose (colocation = 94.1 vs mental-health = 109.2, P = .059). The most common medical disorders in the colocation group were related to metabolic syndrome. CONCLUSIONS: Colocated primary care services were allocated on the basis of severity of psychiatric impairment rather than severity of medical illness. This program serves as a model for other systems to employ for integrated primary and behavioral health services for patients with serious mental illness.

Use of a new alginate film test to study the bactericidal efficacy of the high-level disinfectant ortho-phthalaldehyde.

OBJECTIVES: To evaluate the merit of a new alginate efficacy film test to determine the bactericidal activity of the high-level disinfectant ortho-phthalaldehyde (OPA). METHODS: The efficacy of OPA was investigated using a new sodium alginate surface film test against Mycobacterium chelonae NCIMB 1474 and Epping, and Pseudomonas aeruginosa NCIMB 10421 under different test conditions. RESULTS: OPA was highly bactericidal against P. aeruginosa but its mycobactericidal efficacy was seriously reduced and produced >or=5 log reductions only at a concentration of 0.5% (w/v) within 30-60 min without organic load. CONCLUSIONS: The sodium alginate film efficacy was reproducible between repeats. Inactivation results depended upon the concentration of OPA, contact time, the presence of an organic load and the bacterial genera.

Characterization of peroxisomal 3-hydroxy-3-methylglutaryl coenzyme A reductase in UT2 cells: sterol biosynthesis, phosphorylation, degradation, and statin inhibition.

We have previously identified a CHO cell line (UT2 cells) that expresses only one 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase protein which is localized exclusively in peroxisomes [Engfelt, H.W., Shackelford, J.E., Aboushadi, N., Jessani, N., Masuda, K., Paton, V.G., Keller, G.A., and Krisans, S.K. (1997) J. Biol. Chem. 272, 24579-24587]. In this study, we utilized the UT2 cells to determine the properties of the peroxisomal reductase independent of the endoplasmic reticulum (ER) HMG-CoA reductase. We demonstrated major differences between the two proteins. The peroxisomal reductase is not the rate-limiting enzyme for cholesterol biosynthesis in UT2 cells. The peroxisomal reductase protein is not phosphorylated, and its activity is not altered in the presence of inhibitors of cellular phosphatases. Its rate of degradation is not accelerated in response to mevalonate. Finally, the degradation process is not blocked by N-acetyl-Leu-Leu-norleucinal (ALLN). Furthermore, the peroxisomal HMG-CoA reductase is significantly more resistant to inhibition by statins. Taken together, the data support the conclusion that the peroxisomal reductase is functionally and structurally different from the ER HMG-CoA reductase.

Patient and physician agreement on abdominal pain severity and need for opioid analgesia.

Whereas controversy surrounds emergency department (ED) analgesia administration to patients with undifferentiated abdominal pain, few studies have addressed the level of patient-physician agreement on abdominal pain severity and need for opioid analgesia. This prospective study was undertaken to assess concordance between emergency physicians and patients on abdominal pain severity. Study subjects were a convenience sample of 30 adults seen in an urban university-affiliated tertiary care ED (annual census 65,000) who had undifferentiated abdominal pain meeting an initial severity threshold of 5 on a 10 cm visual analog scale (VAS) marked by the patient. Patients' and physicians' VAS scores, obtained in blinded fashion at presentation (t0) and at one (t1) and two (t2) hours into the ED stay, were compared with t test (VAS scores) and sign-rank (percent change in VAS scores) analyses. In addition, patients and physicians were asked at each assessment time, in blinded fashion, "Is the pain severe enough to warrant morphine?" The kappa statistic was used to characterize the degree of agreement between physician and patient assessments as to whether opioids were indicated. At t0, t1, and t2, patients' mean VAS scores (7.5, 6.7, and 5.1) were significantly (P < .05) higher than the corresponding physicians' VAS scores (5.3, 4.7, and 3.9). Though VAS scores for physicians started lower than those of patients, the percentage changes in scores from one assessment to the next were similar by Wilcoxon sign-rank testing (P > .50 for time intervals t0 - t1 and t1 - t2). Overall, patients and physicians agreed on the question of whether pain was sufficient to warrant opioids in 71 of 90 (78.9%) assessments; the corresponding kappa statistic of .57 indicated moderate agreement (P < .0001). These results, indicating that patients and physicians usually agree on whether opioids are warranted for abdominal pain, have important implications for further research on ED analgesia in this population.

Documenting the factual and the artifactual: Ole Worm and public knowledge.

During the course of his life, Worm made an epistemological shift from the sometimes secretive world of the late Renaissance Neoplatonist and Paracelsian philosopher, from the chemical laborant protecting proprietary processes, to the more open collection of natural and artificial particulars, objects that were stabile, public authorities, regardless of the theories that human minds might spin.

Sex differences in heat pain thresholds as a function of assessment method and rate of rise.

Studies of sex differences in the responses to experimentally induced pain demonstrate greater pain sensitivity among females than males. However, studies investigating heat pain responses have produced inconsistent results. Differences in stimulus characteristics and assessment methods probably account for this variability. This study examined sex differences in the heat pain threshold as a function of two different assessment methods and varying rates of rise. Nineteen female and 18 male healthy volunteers underwent heat pain threshold assessment via the method of levels and the method of limits. In addition, both fast (4.0 degrees C/s) and slow (0.5 degrees C/s) rates of rise were used for the method of levels assessments. In order to examine the reliability of threshold values, each subject participated in two sessions, separated by approximately 8 days. Females evinced lower thresholds than males for the method of levels assessments with both slow and fast rates of rise (ps < 0.05), while no sex differences emerged for the threshold assessed via the method of limits. Test-retest reliability coefficients were relatively high. However, thresholds generally increased significantly from session 1 to session 2. Between method correlations were generally low to moderate. These findings indicate that the method of levels may be more sensitive to sex differences than the more commonly used method of limits. Also, thresholds appear to increase from session 1 to session 2, and thresholds assessed via different methods are not strongly correlated. Potential implications of these results for experimental pain assessment are discussed.

Measuring productivity using RBRVS cost accounting.

The use of a resource-based relative value scale (RBRVS) allows procedures and costs to be indexed on a common or relative basis, using relative value units (RVUs) linked to the amount of resources consumed. Group practice administrators using an RBRVS to account for costs associated with providing services and relating practice revenue and costs to RVUs rather than to fee schedules may find it easier to discuss productivity--and ultimately compensation--with physicians and allied providers. The advantage of RBRVS cost accounting is that it provides a convenient format for comparing physician performance across specialty lines within a group and with external physician groups.

Characterization of UT2 cells. The induction of peroxisomal 3-hydroxy-3-methylglutaryl-coenzyme a reductase.

In the liver 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase is present not only in the endoplasmic reticulum but also in the peroxisomes. However, to date no information is available regarding the function of the peroxisomal HMG-CoA reductase in cholesterol/isoprenoid metabolism, and the structure of the peroxisomal HMG-CoA reductase has yet to be determined. We have identified a mammalian cell line that expresses only one HMG-CoA reductase protein and that is localized exclusively to peroxisomes. This cell line was obtained by growing UT2 cells (which lack the endoplasmic reticulum HMG-CoA reductase) in the absence of mevalonate. The cells exhibited a marked increase in a 90-kDa HMG-CoA reductase that was localized exclusively to peroxisomes. The wild type Chinese hamster ovary cells contain two HMG-CoA reductase proteins, the well characterized 97-kDa protein, localized in the endoplasmic reticulum, and a 90-kDa protein localized in peroxisomes. The UT2 cells grown in the absence of mevalonate containing the up-regulated peroxisomal HMG-CoA reductase are designated UT2*. A detailed characterization and analysis of this cell line is presented in this study.

Cloning and subcellular localization of hamster and rat isopentenyl diphosphate dimethylallyl diphosphate isomerase. A PTS1 motif targets the enzyme to peroxisomes.

To date, isopentenyl diphosphate:dimethylallyl diphosphate isomerase (IPP isomerase; EC 5.3.3.2) is presumed to have a cytosolic localization. However, we have recently shown that in permeabilized cells lacking cytosolic components, mevalonate can be converted to cholesterol, implying that all of the enzymes required for the conversion of mevalonate to farnesyl diphosphate are found in the peroxisome. To provide unequivocal evidence for the subcellular localization of IPP isomerase, in this study, we have cloned the rat and hamster homologues of IPP isomerase and identified the signal that targets this enzyme to peroxisomes. In addition, we also demonstrate that IPP isomerase is regulated at the mRNA level.

Metabolism of farnesol: phosphorylation of farnesol by rat liver microsomal and peroxisomal fractions.

In this study we provide evidence for the first time that rat liver microsomal and peroxisomal fractions are able to phosphorylate free farnesol to its diphosphate ester in a CTP dependent manner. The farnesyl diphosphate (FPP) kinase activity is decreased in whole liver homogenates obtained from rats treated with cholesterol and unchanged in homogenates obtained from rats treated with cholestyramine. In contrast, farnesyl pyrophosphatase (FPPase) activity, an enzyme which specifically hydrolyzes FPP to farnesol is only found in the microsomal fraction and is unaffected by treatment of rats with cholesterol or cholestyramine. In addition, we also demonstrate that farnesol can be oxidized to a prenyl aldehyde, presumably by an alcohol dehydrogenase (ADH), and that this activity resides in the mitochondrial and peroxisomal fractions.

Corrosion at the interface. A possible solution to cobalt-chrome heads on titanium alloy stems.

The low-wear characteristics of cobalt-chrome femoral heads matched with the excellent biocompatibility and low modulus of titanium alloy femoral stems constitute the preferred combination used by many orthopaedic surgeons performing total hip arthroplasty. The combination of these materials in a synovial fluid environment, however, has proven to result in extensive crevice corrosion and metallosis of the surrounding tissues. This study investigates an alternative to the conventional mating of dissimilar metals at the head-neck junction. Five cobalt-chrome heads premated with titanium alloy sleeves were investigated by gross examination, dissecting microscopy, and scanning electron microscopy. Examination by both gross examination and dissecting microscope revealed no signs of corrosion. Scanning electron microscope examination revealed slight crevice corrosion in the only head with a +15-mm neck length.

Mevalonate kinase is predominantly localized in peroxisomes and is defective in patients with peroxisome deficiency disorders.

We reported recently that mevalonate kinase (EC 2.7.1.36; ATP:mevalonate 5-phosphotransferase) that was isolated from rat liver and believed to be a cytosolic protein was localized in rat liver peroxisomes. In addition, we found that the mevalonate kinase monoclonal antibody used in the study also reacted with several other proteins present in the mitochondrial and cytosolic fractions. These findings raised the prospect of the presence of several isoenzymes of mevalonate kinase localized in different compartments of the cell. In the current study we produced four new polyclonal antibodies against different epitopes of mevalonate kinase to investigate the subcellular localization of the protein by several different approaches: (i) by analytical subcellular fractionation and immunoblotting of mevalonate kinase in the isolated subcellular fractions with the monospecific antibodies; (ii) by immunocryoelectron microscopy techniques; and (iii) by expressing the cDNA encoding mevalonate kinase in mammalian cells. The data obtained demonstrate that there is only one mevalonate kinase protein that is predominantly localized in peroxisomes. We also illustrate that the protein is targeted to and imported into peroxisomes. In addition, we show that in cells and tissues obtained from patients with peroxisomal deficiency diseases mevalonate kinase protein and its activity are severely reduced.

Subcellular localization of squalene synthase in rat hepatic cells. Biochemical and immunochemical evidence.

In the present study we investigated the subcellular localization of squalene synthase (farnesyl-diphosphate:farnesyl-diphosphate farnesyltransferase, EC 2.5.1.21). Squalene synthase catalyzes the formation of squalene from trans-farnesyl diphosphate in two distinct steps and is the first committed enzyme for the biosynthesis of cholesterol. Recently, a truncated form of the enzyme from rat hepatocytes was purified, and monospecific antibodies for squalene synthase were produced. This enabled the subcellular localization of squalene synthase by three different methods: (i) analytical subcellular fractionation and measurements of enzyme activities; (ii) immunodeterminations of squalene synthase in the isolated subcellular fractions with a monospecific antibody; and (iii) immunoelectron microscopy. All three methods gave consistent results. The data clearly illustrate that squalene synthase enzymatic activity and squalene synthase are exclusively localized in the endoplasmic reticulum. In rat hepatic peroxisomes we were not able to detect any squalene synthase. In addition, we also demonstrated that squalene synthase in the microsomal fraction is dramatically regulated by a number of hypolipidemic drugs and dietary treatments.

Mevalonate kinase is localized in rat liver peroxisomes.

Recent data suggest that rat liver peroxisomes play a critical role in cholesterol synthesis. Specifically, peroxisomes contain a number of enzymes required for cholesterol synthesis as well as sterol carrier protein-2. Furthermore, peroxisomes are involved in the in vitro synthesis of cholesterol from mevalonate and contain significant levels of apolipoprotein E, a major constituent of several classes of plasma lipoproteins. In this study we have investigated the subcellular localization of mevalonate kinase (EC 2.7.1.36; ATP:mevalonate-5-phosphotransferase). Mevalonate kinase is believed to be a cytosolic enzyme and catalyzes the phosphorylation of mevalonate to form mevalonate 5-phosphate. Mevalonate kinase has been purified from rat liver cytosol and a cDNA clone coding for rat mevalonate kinase has also been isolated and characterized. In this study, utilizing monoclonal antibodies made against the purified rat mevalonate kinase, we demonstrate the presence of mevalonate kinase in rat liver peroxisomes and in the cytosol. Each of these compartments contained a different form of the protein. The pI and the Mr of the peroxisomal protein is 6.2 and 42,000, respectively. The pI and Mr of the cytosolic protein is 6.9 and 40,000, respectively. The peroxisomal protein was also significantly induced by a number of different hypolipidemic drugs. In addition, we present evidence for the unexpected finding that the purified mevalonate kinase (isolated from the cytosol and assumed to be a cytosolic protein) is actually a peroxisomal protein.

Synthesis and secretion of serum gelsolin by smooth muscle tissue.

Gelsolin is one of many actin binding proteins which regulate the structure of intracellular microfilaments. A secretory form of gelsolin, a protein also known as "actin depolymerizing factor" or "brevin," is present in animal sera. In the present studies, we: demonstrate that a 90-kDa secretory protein produced by chicken gizzard smooth muscle is serum gelsolin; show that chicken serum gelsolin, as compared with its mammalian counterparts, lacks 26 amino acid residues at its NH2-terminal end; show that gizzard smooth muscle devotes on the order of 100 times more of its total protein synthetic effort (about 1% of the total) to the production of serum gelsolin than does liver, a previously speculated major source of this protein; and give evidence that rat tissues which are rich in smooth muscle cells (blood vessels, uterine muscle) also produce serum gelsolin. Our work suggests that, in vivo, smooth muscle-containing tissues may be major producers of the serum form of this actin binding protein.

Specific proteolytic modification of creatine kinase isoenzymes. Implication of C-terminal involvement in enzymic activity but not in subunit-subunit recognition.

We are using the isoenzymes of creatine kinase (CK) to investigate the effect of specific proteolytic modification on the abilities of enzyme subunits to establish precise subunit-subunit recognition in vitro. Previous work by others has shown that treatment of the MM isoenzyme of rabbit CK with Proteinase K results in a specific proteolytic modification and inactivation of the enzyme. In the present work, we show that both the MM and BB isoenzymes of chicken CK are also specifically modified by Proteinase K, resulting in over 98% loss of catalytic activity and approx. 10% decreases in subunit molecular masses of the enzymes. Similar reactions appear to occur when the isoenzymes are treated with Pronase E. Limited amino acid sequence analysis of intact and Proteinase K-modified MM-CK suggests that the proteolytic modification results from a single peptide-bond cleavage occurring between alanine residues 328 and 329, about 50 amino acid residues from the C-terminal end; the active-site cysteine residue was recovered in the large protein fragment of modified M-CK subunits. Proteolytically modified M-CK and B-CK subunits were able to refold and reassociate into dimeric structures after treatment with high concentrations of LiCl and at low pH. Thus the proteolytically modified CK subunits retain their ability to refold and to establish precise subunit-subunit recognition in vitro.

Synthesis of apolipoprotein A1 in skeletal muscles of normal and dystrophic chickens.

We recently observed that, around the time of hatching, chick skeletal muscles synthesize and secrete apolipoprotein A1 (apo-A1) at high rates and that reinitiation of synthesis of this serum protein to high levels occurs in mature chicken breast muscle following surgical denervation (Shackelford, J. E., and Lebherz, H. G. (1983) J. Biol. Chem. 258, 7175-7180; 14829-14833). In the present work we investigate the effect of avian muscular dystrophy on the synthesis of apo-A1 in chicken muscles. The relative rate of synthesis of apo-A1 and levels of apo-A1 RNA in mature dystrophic breast (fast-twitch) muscle were about 6-fold higher than normal, while synthesis of apo-A1 in breast muscles derived from 2-day-old dystrophic chicks was close to normal. These observations suggest that the elevated apo-A1 synthetic rate in mature dystrophic breast muscle results from a failure of the diseased tissue to "shut down" apo-A1 synthesis to the normal level during postembryonic maturation. Apo-A1 synthesis in the "slow-twitch" lateral adductor muscle of dystrophic chickens was found to be normal. Our work is discussed in terms of the apparent similarities between the effects of surgical denervation and muscular dystrophy on the protein synthetic programs expressed by chicken skeletal muscles.