Periodic venting of MABR lumen allows high removal rates and high gas-transfer efficiencies.

The membrane-aerated biofilm reactor (MABR) is a novel treatment technology that employs gas-supplying membranes to deliver oxygen directly to a biofilm growing on the membrane surface. When operated with closed-end membranes, the MABR provides 100-percent oxygen transfer efficiencies (OTE), resulting in significant energy savings. However, closed-end MABRs are more sensitive to back-diffusion of inert gases, such as nitrogen. Back-diffusion reduces the average oxygen transfer rates (OTR), consequently decreasing the average contaminant removal fluxes (J). We hypothesized that venting the membrane lumen periodically would increase the OTR and J. Using an experimental flow cell and mathematical modeling, we showed that back-diffusion gas profiles developed over relatively long timescales. Thus, very short ventings could re-establish uniform gas profiles for relatively long time periods. Using modeling, we systematically explored the effect of the venting interval (time between ventings). At moderate venting intervals, opening the membrane for 20 s every 30 min, the venting significantly increased the average OTR and J without substantially impacting the OTEs. When the interval was short enough, in this case shorter than 20 min, the OTR was actually higher than for continuous open-end operation. Our results show that periodic venting is a promising strategy to combine the advantages of open-end and closed end operation, maximizing both the OTR and OTE.

The new kidney disease: improving global outcomes (KDIGO) guidelines - expert clinical focus on bone and vascular calcification.

Chronic kidney disease-mineral and bone disorder (CKD-MBD) defines a triad of interrelated abnormalities of serum biochemistry, bone and the vasculature associated with chronic kidney disease (CKD). The new kidney disease: improving global outcomes (KDIGO) guidelines define the quality and depth of evidence supporting therapeutic intervention in CKD-MBD. They also highlight where patient management decisions lack a strong evidence base. Expert interpretation of the guidelines, along with informed opinion, where evidence is weak, may help develop effective clinical practice. The body of evidence linking poor bone health and reservoir function (the ability of bone to buffer calcium and phosphorus) with vascular calcification and cardiovascular outcomes is growing. Treating renal bone disease should be one of the primary aims of therapy for CKD. Evaluation of the biochemical parameters of CKD-MBD (primarily phosphorus, calcium, parathyroid hormone and vitamin D levels) as early as CKD Stage 3, and an assessment of bone status (by the best means available), should be used to guide treatment decisions. The adverse effects of high phosphorus intake relative to renal clearance (including stimulation of hyperparathyroidism) precede hyperphosphatemia, which presents late in CKD. Early reduction of phosphorus load may ameliorate these adverse effects. Evidence that calcium load may influence progression of vascular calcification with effects on mortality should also be considered when choosing the type and dose of phosphate binder to be used. The risks, benefits, and strength of evidence for various treatment options for the abnormalities of CKD-MBD are considered.

Evidence of specialized bromate-reducing bacteria in a hollow fiber membrane biofilm reactor.

Bromate is a carcinogenic disinfection by-product formed from bromide during ozonation or advanced oxidation. We previously observed bromate reduction in a hydrogen-based, denitrifying hollow fiber membrane biofilm reactor (MBfR). In this research, we investigated the potential existence of specialized bromate-reducing bacteria. Using denaturing gradient gel electrophoresis (DGGE), we compared the microbial ecology of two denitrifying MBfRs, one amended with nitrate as the electron acceptor and the other with nitrate plus bromate. The DGGE results showed that bromate exerted a selective pressure for a putative, specialized bromate-reducing bacterium, which developed a strong presence only in the reactor with bromate. To gain further insight into the capabilities of specialized, bromate-reducing bacteria, we explored bromate reduction in a control MBfR without any primary electron acceptors. A grown biofilm in the control MBfR reduced bromate without previous exposure, but the rate of reduction decreased over time, especially after perturbations resulting in biomass loss. The decrease in bromate reduction may have been the result of the toxic effects of bromate. We also used batch tests of the perchlorate-reducing pure culture, Dechloromonas sp. PC1 to test bromate reduction and growth. Bromate was reduced without measurable growth. Based on these results, we speculate bromate's selective pressure for the putative, specialized BRB observed in the DGGE was not growth related, but possibly based on resistance to bromate toxicity.

Detection of cognitive decline after coronary surgery: a comparison of computerized and conventional tests.

BACKGROUND: Postoperative cognitive decline is a common complication after coronary artery bypass graft (CABG) surgery. Postoperative cognitive decline is defined on the basis of change in cognitive function detected with repeated assessments using neuropsychological tests. Therefore improvement in neuropsychological testing instruments may increase our understanding of postoperative cognitive decline. METHODS: Fifty patients undergoing CABG surgery completed both a conventional and a computerized battery of tests before and 6 days after CABG surgery. Fifty age- and education-matched controls completed the same test batteries 6 days apart. The reliability and the sensitivity to postoperative cognitive decline were computed for each battery. RESULTS: Both test batteries detected postoperative cognitive decline 6 days after CABG surgery. For the computerized battery, the reliability of the reaction times (intraclass correlation 0.89-0.92) was greater than for any test from the conventional battery (intraclass correlation 0.56-0.71), although accuracy measures were less reliable (intraclass correlation 0.61-0.89). The computerized battery detected all the cases of POCD identified by the conventional test battery and also five cases that were classified as normal by the conventional tests. CONCLUSION: Computerized tests are suitable for measuring cognitive change after CABG surgery and may detect change in a greater proportion of patients 6 days after CABG surgery than conventional neuropsychological tests.

Mechanism of retinoic acid induced attenuation of PTH action in UMR 106-01 cells.

Studies from our laboratory in osteoblast-like cells have shown that the increase in EGF receptor expression in response to PTH was cyclic AMP mediated and was blocked by treatment with retinoic acid (RA). The present studies investigate the mechanism for this effect of RA on PTH actions. UMR 106-01 cells were exposed to RA and were tested for cAMP response to PTH as well as for (125)I PTH binding. cAMP production in response to PTH was markedly decreased by RA (25.1 +/- 1.6% of control) whereas there was only a slight decrease in PTH binding in response to RA. For the study of adenylate cyclase activity, membranes were isolated from intact cells that had been exposed to RA. Treatment with RA decreased PTH-stimulated adenylate cyclase activity; however, forskolin-stimulated enzyme activity was unchanged. Treatment of intact cells with pertussis toxin, to inactivate Gi, did not alter the inhibitory effect of RA on PTH-stimulated adenylate cyclase activity. Addition of GppNHp, a non-hydrolyzable analogue of GTP, completely restored the response to PTH in the membranes. Therefore, we examined the activity of IMP dehydrogenase, the rate-limiting enzyme for GTP biosynthesis, and GMP reductase which counteracts the effect of the synthetic enzyme. Treatment with RA for 48 hours increased GMP reductase activity by 240.9 +/- 24.2% and decreased IMP dehydrogenase activity to 67.5 +/- 8.8% of control values. These data indicate that RA impairs the response to PTH in intact cells. This blunted response was preserved in membrane preparations but was corrected by GTP. The RA-induced alterations of enzymes involved in the GTP biosynthetic pathway in a direction that favors a decrease in GTP biosynthesis provide an explanation for the inhibitory effect of RA on PTH actions.

Site-specific phosphorylation of human p53 protein determined by mass spectrometry.

Human recombinant p53 (r-p53) protein was studied by mass spectrometry (MS) to determine site-specific posttranslational differences between basal and hyperphosphorylated r-p53. Wild-type p53 was basally expressed after baculovirus infection while a parallel preparation was treated with the phosphatase inhibitor okadaic acid during the terminal stages of expression to create a hyperphosphorylated form of p53 known for its higher DNA binding and transcriptional activation. After immunoaffinity and HPLC purification, MALDI/MS measured a higher molecular mass for r-p53 from okadaic acid treatment relative to control, suggesting a higher phosphorylation state. This was supported by an acidic shift of r-p53 isoforms separated by gel isoelectric focusing. Employing a variety of mass spectrometric analyses combined with separation and affinity techniques, six specific phosphorylation sites of p53 were identified. The MS data indicated that hyperphosphorylated p53 showed a higher degree of phosphorylation than basal p53 at specific amino- and carboxy-terminal sites. In particular, ESI-MS demonstrated that Ser(315) was entirely phosphorylated after okadaic acid treatment, as confirmed biochemically by CDK2 kinase assay and by isoelectric focusing. In summary, MS analysis uniquely revealed increased, site-specific phosphorylations on p53 after phosphatase inhibition, particularly at Ser(315), which may be critical molecular events in defining p53 activity.

High-sensitivity array analysis of gene expression for the early detection of disseminated breast tumor cells in peripheral blood.

Early detection is an effective means of reducing cancer mortality. Here, we describe a highly sensitive high-throughput screen that can identify panels of markers for the early detection of solid tumor cells disseminated in peripheral blood. The method is a two-step combination of differential display and high-sensitivity cDNA arrays. In a primary screen, differential display identified 170 candidate marker genes differentially expressed between breast tumor cells and normal breast epithelial cells. In a secondary screen, high-sensitivity arrays assessed expression levels of these genes in 48 blood samples, 22 from healthy volunteers and 26 from breast cancer patients. Cluster analysis identified a group of 12 genes that were elevated in the blood of cancer patients. Permutation analysis of individual genes defined five core genes (P < or = 0.05, permax test). As a group, the 12 genes generally distinguished accurately between healthy volunteers and patients with breast cancer. Mean expression levels of the 12 genes were elevated in 77% (10 of 13) untreated invasive cancer patients, whereas cluster analysis correctly classified volunteers and patients (P = 0.0022, Fisher's exact test). Quantitative real-time PCR confirmed array results and indicated that the sensitivity of the assay (1:2 x 10(8) transcripts) was sufficient to detect disseminated solid tumor cells in blood. Expression-based blood assays developed with the screening approach described here have the potential to detect and classify solid tumor cells originating from virtually any primary site in the body.

Inactivation of DNA mismatch repair by increased expression of yeast MLH1.

Inactivation of DNA mismatch repair by mutation or by transcriptional silencing of the MLH1 gene results in genome instability and cancer predisposition. We recently found (P. V. Shcherbakova and T. A. Kunkel, Mol. Cell. Biol. 19:3177-3183, 1999) that an elevated spontaneous mutation rate can also result from increased expression of yeast MLH1. Here we investigate the mechanism of this mutator effect. Hybridization of poly(A)(+) mRNA to DNA microarrays containing 96.4% of yeast open reading frames revealed that MLH1 overexpression did not induce changes in expression of other genes involved in DNA replication or repair. MLH1 overexpression strongly enhanced spontaneous mutagenesis in yeast strains with defects in the 3'-->5' exonuclease activity of replicative DNA polymerases delta and epsilon but did not enhance the mutation rate in strains with deletions of MSH2, MLH1, or PMS1. This suggests that overexpression of MLH1 inactivates mismatch repair of replication errors. Overexpression of the PMS1 gene alone caused a moderate increase in the mutation rate and strongly suppressed the mutator effect caused by MLH1 overexpression. The mutator effect was also reduced by a missense mutation in the MLH1 gene that disrupted Mlh1p-Pms1p interaction. Analytical ultracentrifugation experiments showed that purified Mlh1p forms a homodimer in solution, albeit with a K(d) of 3.14 microM, 36-fold higher than that for Mlh1p-Pms1p heterodimerization. These observations suggest that the mismatch repair defect in cells overexpressing MLH1 results from an imbalance in the levels of Mlh1p and Pms1p and that this imbalance might lead to formation of nonfunctional mismatch repair complexes containing Mlh1p homodimers.

The effect of vitamin E and vitamin C supplementation on LDL oxidizability and neutrophil respiratory burst in young smokers.

OBJECTIVE: The purpose of this study was to determine the effect of vitamin E and/or vitamin C supplementation on low-density lipoprotein (LDL) oxidizability and neutrophil (PMN) superoxide anion production in young smokers. METHODS: Thirty smokers with a <5 pack-year history were randomly assigned to take placebo; vitamin C (1 g/day); vitamin E (400 IU/day), or both vitamins in a double-blind fashion. Subjects took the supplements for 8 weeks. At weeks 0 and 8, blood was collected for isolation of LDL and PMN, and for antioxidant vitamin analysis. LDL was oxidized with a copper (Cu) catalyst, and oxidation was measured by formation of conjugated dienes over a 5-hour time course. Lag times and maximum oxidation rates were calculated from the time course data. PMN superoxide anion release was assessed by respiratory burst after stimulation with phorbol ester and opsonized zymosan, and their ability to oxidize autologous LDL following treatment with the above stimuli was measured with the conjugated diene assay. RESULTS: Subjects who received vitamin E alone had a significant increase in the lag phase of Cu-catalyzed LDL oxidation (week 0, 118+/-31 min vs. week 8, 193+/-80 min, mean +/- SD, p < 0.05), whereas the vitamin C and placebo groups had no changes in LDL oxidation kinetics. The group receiving both vitamins E and C had a significant reduction in oxidation rate (week 0. 7.4+/-2.3 vs. week 8, 5.1+/-2.1, p < 0.05). There were no significant changes for any group in PMN superoxide anion production or PMN LDL oxidation after stimulation with either phorbol ester or opsonized zymosan. Plasma and LDL vitamin E concentrations were significantly increased in both groups that received vitamin E. The subjects who received vitamin C alone had no significant change in plasma vitamin C concentrations; however, when data were pooled from both groups who received vitamin C, the increases were significant. CONCLUSION: Vitamin E supplementation of young smokers was effective in reducing Cu-catalyzed LDL oxidizability; however, vitamin E and/or C supplementation showed few significant effects on the more physiologically relevant PMN function. This casts doubt on the ability of antioxidant supplementation to reduce oxidative stress in smokers in vivo. Therefore, smoking cessation remains the only means by which young smokers can prevent premature coronary heart disease.

Linking gene expression patterns to therapeutic groups in breast cancer.

A major objective of current cancer research is to develop a detailed molecular characterization of tumor cells and tissues that is linked to clinical information. Toward this end, we have identified approximately one-quarter of all genes that were aberrantly expressed in a breast cancer cell line using differential display. The cancer cells lost the expression of many genes involved in cell adhesion, communication, and maintenance of cell shape, while they gained the expression of many synthetic and metabolic enzymes important for cell proliferation. High-density, membrane-based hybridization arrays were used to study mRNA expression patterns of these genes in cultured cells and archived tumor tissue. Cluster analysis was then used to identify groups of genes, the expression patterns of which correlated with clinical information. Two clusters of genes, represented by p53 and maspin, had expression patterns that strongly associated with estrogen receptor status. A third cluster that included HSP-90 tended to be associated with clinical tumor stage, whereas a forth cluster that included keratin 14 tended to be associated with tumor size. Expression levels of these clinically relevant gene clusters allowed breast tumors to be grouped into distinct categories. Gene expression fingerprints that include these four gene clusters have the potential to improve prognostic accuracy and therapeutic outcomes for breast cancer patients.

Down-regulation of laminin-5 in breast carcinoma cells.

BACKGROUND: Laminin-5 (ln-5), a large heterotrimeric glycoprotein consisting of an alpha 3, beta 3, and gamma 2 chain, is a component of epithelial cell basement membranes that functions as a ligand of the alpha 3 beta 1 and alpha 6 beta 4 integrins to regulate cell adhesion, migration, and morphogenesis. The ln-5 chains show tissue-specific patterns of regulation in tumors derived from different tissues. For example, ln-5 is often up-regulated in gliomas, gastric carcinomas, and squamous carcinomas and down-regulated in prostate and basal cell carcinomas. Ln-5 expression patterns may represent useful tumor markers and help to elucidate the role of ln-5 in tumor progression in different tissue types. MATERIALS AND METHODS: We have studied ln-5 expression patterns in the breast. mRNA levels were examined in tumor and normal breast epithelial cell lines, tissue samples, and immunomagnetically sorted primary cultures using differential display, Northern blotting, and hybridization arrays. Protein levels were examined by immunoprecipitation. Gene integrity was assessed by Southern blotting of representative cell types. RESULTS: Ln-5 alpha 3, beta 3, and gamma 2 mRNA expression was found to be markedly down-regulated in a panel of breast tumor cell lines when compared with normal breast epithelial cells. Ln-5 mRNA was expressed at relatively high levels in MCF-10A immortal normal breast epithelial cells, long-term cultures of normal breast cells, and sorted primary cultures of normal breast luminal epithelial and myoepithelial cells. Reduced, but detectable, levels of ln-5 tended to be expressed in cell lines derived from early-stage breast tumors, whereas expression was generally not detected in cell lines derived from later-stage tumors. In breast tumor tissue specimens, expression of ln alpha 3 and beta 3 mRNAs tended to be reduced relative to levels observed in adjacent nontumor tissue, whereas in gamma 2 levels were elevated in specimens with increased amounts of myoepithelial cells. These ln-5 expression changes could not be attributed to large-scale mutations or gene rearrangements. Ln-5 protein levels were found to reflect mRNA levels in representative cell lines. At senescence, a growth state believed to suppress tumorigenesis, expression of all three ln-5 mRNAs was up-regulated. CONCLUSION: The down-regulation of ln-5 mRNA expression in breast tumors cells provides a new molecular marker and suggests that ln-5 functions to control tumor progression in the breast.

Therapy of secondary hyperparathyroidism with 19-nor-1alpha,25-dihydroxyvitamin D2.

Secondary hyperparathyroidism contributes to significant morbidity in patients with chronic renal failure. The treatment of this disorder with vitamin D compounds, such as calcitriol, although effective at suppressing parathyroid hormone (PTH) secretion, may promote the development of hypercalcemia and hyperphosphatemia, thus increasing the risk for metastatic calcification. A new vitamin D analogue, 19-nor-1alpha,25-(OH)2D2 (paricalcitol; Zemplar, Abbott Laboratories, Inc, Chicago, IL) has recently been developed for the treatment of secondary hyperparathyroidism, and, in experimental animals, it was found to be less calcemic and phosphatemic than calcitriol. In double-blind clinical trials, paricalcitol effectively decreased the levels of PTH by 60%, yet the mean serum calcium values remained within the normal range. The few episodes of hypercalcemia that occurred in the paricalcitol-treated patients (8 of 400 determinations > or =11.0 mg/dL in 7 patients) were associated with marked decreases in PTH levels (87% +/- 2% less than baseline) and absolute values of PTH less than 100 pg/mL in four of the seven patients. PTH values less than 100 pg/mL, however, occurred in 15 patients, but were not invariably associated with frank hypercalcemia, although serum calcium levels increased to 10.63 +/- 0.3 mg/dL, slightly greater than the upper limits of normal. Additional studies to evaluate the conversion from calcitriol to paricalcitol therapy showed that a dose ratio of 1:4 (calcitriol:paricalcitol) could maintain control of high levels of PTH without significant alterations in serum calcium and phosphorus levels. These studies indicate that effective control of hyperparathyroidism can be achieved with paricalcitol therapy with minimal perturbation of serum calcium and phosphorus levels, and may have a therapeutic advantage over current treatment strategies.

Terminal differentiation of human breast cancer through PPAR gamma.

We have previously demonstrated that PPAR gamma stimulates the terminal differentiation of adipocyte precursors when activated by synthetic ligands, such as the antidiabetic thiazolidinedione (TZD) drugs. We show here that PPAR gamma is expressed at significant levels in human primary and metastatic breast adenocarcinomas. Ligand activation of this receptor in cultured breast cancer cells causes extensive lipid accumulation, changes in breast epithelial gene expression associated with a more differentiated, less malignant state, and a reduction in growth rate and clonogenic capacity of the cells. Inhibition of MAP kinase, shown previously to be a powerful negative regulator of PPAR gamma, improves the TZD ligand sensitivity of nonresponsive cells. These data suggest that the PPAR gamma transcriptional pathway can induce terminal differentiation of malignant breast epithelial cells and thus may provide a novel, nontoxic therapy for human breast cancer.

Identification and verification of differential display cDNAs using gene-specific primers and hybridization arrays.

An accurate and streamlined approach to differential display (DD) band identification and verification is described. To minimize false positives, the strategy avoids the use of impure Northern blot probes obtained from PCR-amplified DD bands. To increase throughput, the cloning of DD bands is replaced by a gene-specific primer approach, and hybridization arrays are used in place of Northern blots. In summary, DD bands obtained with long primers were directly sequenced to allow the design and synthesis of gene-specific primers, which were then used to PCR-amplify homogeneous probes for the verification of expression patterns by hybridization array analysis. Differential expression of 60 of the 63 genes tested was confirmed. Thus, false positives are not inherent to DD. The results demonstrate the power of DD used with hybridization arrays to rapidly generate information on expression patterns of differentially expressed genes.

Expression genetics: a different approach to cancer diagnosis and prognosis.

Expression genetics is a new approach to the identification of cancer-related genes. Instead of studying gene mutations at the genome level, it focuses on the investigation of heredity at the RNA level. By isolating genes whose expression is up or down regulated in cancers, expression geneticists study their function in the context of gene regulation. A major goal of expression genetics in cancer is to correct gene expression in tumors by the application of potential therapeutic agents.

Retinoids modulate the effect of PTH and calcitriol on EGF receptor expression in UMR 106-01 cells.

Although osteoblast proliferation is a prominent feature of osteitis fibrosa, studies in vitro using osteoblast-like cells have shown that parathyroid hormone (PTH) impairs cell growth. Recent studies in our laboratory have shown that PTH increases epidermal growth factor (EGF) receptor expression in UMR 106-01 osteoblast-like cells, and thus, osteoblast proliferation may occur as a result of an enhanced response of the osteoblast to EGF. In the present studies we investigated the effect of calcitriol and the influence of retinoids on the regulation of EGF receptors. Calcitriol increased 125I-EGF binding 2.5-3-fold after 72 hours of incubation and was maximal at a calcitriol dose of 100 nM. Scatchard analysis showed that this effect was due to increased receptor number. In contrast, all-trans retinoic acid or 9-cis retinoic acid alone, even at 10 microM, caused less than a 50% increase in 125I-EGF binding. However, the effect of calcitriol was totally abolished in the presence of all-trans retinoic acid. 9-cis retinoic acid was equivalent with all-trans retinoic acid in this regard. In the presence of either retinoid, the stimulatory effect of PTH was totally eliminated and EGF binding was actually decreased below control values. Additional studies revealed that retinoic acid decreased PTH-stimulated cAMP generation in a dose-dependent manner. These data are consistent with our previous studies which showed that the effect of PTH on the induction of EGF receptors was mediated by a cAMP-dependent mechanism. The inhibition of the calcitriol effect by retinoids is consistent with the requirement of the retinoid-X-receptor (RXR) for binding of the vitamin D receptor (VDR) to its target sequences in DNA. These data indicate that EGF receptors in UMR 106-01 cells are up-regulated by PTH and calcitriol and that this process can be modulated by retinoids. Retinoids, therefore, may play a major role in the regulation of osteoblast function by PTH and calcitriol.

Creatine and phosphocreatine analogs: anticancer activity and enzymatic analysis.

The brain isoform of creatine kinase has been implicated in cellular transformation processes. Cyclocreatine, a creatine kinase substrate analog, was previously shown to be cytotoxic to a broad spectrum of solid tumors. We have synthesized, enzymatically characterized, and evaluated the antitumor activity of a series of substrate analogs of creatine kinase. Using in vitro assays, we demonstrate that several of these analogs are cytotoxic to the human ME-180 cervical carcinoma, the MCF-7 breast adenocarcinoma and the HT-29 colon adenocarcinoma cell lines at low mM concentrations. Analogs that were active in vitro delayed the growth of a subcutaneously implanted rat 13,762 mammary adenocarcinoma. Tumor growth delays of 6-8 days were achieved, which is comparable to effects seen with standard regimens of currently used anticancer drugs. These studies further establish the creatine kinase system as a promising and novel target for anticancer chemotherapy drug design.