Focal status epilepticus and progressive dyskinesia: A novel phenotype for glycine receptor antibody-mediated neurological disease in children.

BACKGROUND: Antibody-associated disorders of the central nervous system are increasingly recognised in adults and children. Some are known to be paraneoplastic, whereas in others an infective trigger is postulated. They include disorders associated with antibodies to N-methyl-d-aspartate receptor (NMDAR), voltage-gated potassium channel-complexes (VGKC-complex), GABAB receptor or glycine receptor (GlyR). With antibodies to NMDAR or VGKC-complexes, distinct clinical patterns are well characterised, but as more antibodies are discovered, the spectra of associated disorders are evolving. GlyR antibodies have been detected in patients with progressive encephalopathy with rigidity and myoclonus (PERM), or stiff man syndrome, both rare but disabling conditions. CASE REPORT: We report a case of a young child with focal seizures and progressive dyskinesia in whom GlyR antibodies were detected. Anticonvulsants and immunotherapy were effective in treating both the seizures and movement disorder with good neurological outcome and with a decline in the patient's serum GlyR-Ab titres. CONCLUSION: Glycine receptor antibodies are associated with focal status epilepticus and seizures, encephalopathy and progressive dyskinesia and should be evaluated in autoimmune encephalitis.

DLX1 acts as a crucial target of FOXM1 to promote ovarian cancer aggressiveness by enhancing TGF-ß/SMAD4 signaling.

Recent evidence from a comprehensive genome analysis and functional studies have revealed that FOXM1 is a crucial metastatic regulator that drives cancer progression. However, the regulatory mechanism by which FOXM1 exerts its metastatic functions in cancer cells remains obscure. Here, we report that DLX1 acts as a FOXM1 downstream target, exerting pro-metastatic function in ovarian cancers. Both FOXM1 isoforms (FOXM1B or FOXM1C) could transcriptionally upregulate DLX1 through two conserved binding sites, located at +61 to +69bp downstream (TFBS1) and -675 to -667bp upstream (TFBS2) of the DLX1 promoter, respectively. This regulation was further accentuated by the significant correlation between the nuclear expression of FOXM1 and DLX1 in high-grade serous ovarian cancers. Functionally, the ectopic expression of DLX1 promoted ovarian cancer cell growth, cell migration/invasion and intraperitoneal dissemination of ovarian cancer in mice, whereas small interfering RNA-mediated DLX1 knockdown in FOXM1-overexpressing ovarian cancer cells abrogated these oncogenic capacities. In contrast, depletion of FOXM1 by shRNAi only partially attenuated tumor growth and exerted almost no effect on cell migration/invasion and the intraperitoneal dissemination of DLX1-overexpressing ovarian cancer cells. Furthermore, the mechanistic studies showed that DLX1 positively modulates transforming growth factor-ß (TGF-ß) signaling by upregulating PAI-1 and JUNB through direct interaction with SMAD4 in the nucleus upon TGF-ß1 induction. Taken together, these data strongly suggest that DLX1 has a pivotal role in FOXM1 signaling to promote cancer aggressiveness through intensifying TGF-ß/SMAD4 signaling in high-grade serous ovarian cancer cells.

Oriented polyvinylidene fluoride-trifluoroethylene (P(VDF-TrFE)) films by Langmuir-Blodgett deposition: a synchrotron X-ray diffraction study.

Langmuir-Blodgett films of polyvinylidene fluoride trifluoroethylene - P(VDF-TrFE)-copolymers possess substantially improved electrocaloric and pyroelectric properties, when compared with conventionally spin-cast films. In order to rationalize this, we prepared single-layered films of P(VDF-TrFE) (70 : 30) using both deposition techniques. Grazing incidence wide-angle X-ray scattering (GIWAXS), reveals that Langmuir-Blodgett deposited films have a higher concentration of the ferroelectric ß-phase crystals, and that these films are highly oriented with respect to the substrate. Based on these observations, we suggest alternative means of deposition, which may substantially enhance the electrocaloric effect in P(VDF-TrFE) films. This development has significant implications for the potential use of P(VDF-TrFE) in solid-state refrigeration.

The interaction between C35 and ΔNp73 promotes chemo-resistance in ovarian cancer cells.

BACKGROUND: The purpose of this study was to characterise the oncogenic roles of C35, a novel protein binding partner of ΔNp73, in ovarian cancer and to investigate the functional significance of C35-ΔNp73 interaction in the regulation of chemo-resistance. METHODS: C35 expression was evaluated by quantitative real-time PCR in human ovarian cancer tissues and cell lines. The aggressiveness of ovarian cancer cells overexpressing C35 was examined by cell proliferation, migration, soft agar and nude mouse xenograft. The significance of C35-ΔNp73 interaction in chemo-resistance was evaluated by apoptosis assays and cell viability after cisplatin treatment. RESULTS: The expression of C35 was significantly enhanced in human ovarian cancer tissues. Overexpression of C35 augmented proliferation, migration and tumourigenicity in ovarian cancer cell lines. C35 knockdown inhibited cell motility and cell growth. The co-expression of C35 and ΔNp73 by transient or stable transfection in ovarian cancer cells induced greater resistance to cisplatin treatment than did transfection with C35 or ΔNp73 alone. The cisplatin resistance was demonstrated to be caused by increased AKT and NFκB activity induced by C35-ΔNp73. CONCLUSION: Our results suggest that ΔNp73 might cooperate with C35 to promote tumour progression and contribute to cisplatin resistance in ovarian cancer cells. Future studies of the functional roles of ΔNp73 and C35 will provide insight that will aid in the establishment of new strategies and more effective therapies.

Platform for establishing interlaboratory reproducibility of selected reaction monitoring-based mass spectrometry peptide assays.

Mass spectrometry (MS) is an attractive alternative to quantification of proteins by immunoassays, particularly for protein biomarkers of clinical relevance. Reliable quantification requires that the MS-based assays are robust, selective, and reproducible. Thus, the development of standardized protocols is essential to introduce MS into clinical research laboratories. The aim of this study was to establish a complete workflow for assessing the transferability and reproducibility of selected reaction monitoring (SRM) assays between clinical research laboratories. Four independent laboratories in North America, using identical triple-quadrupole mass spectrometers (Quantum Ultra, Thermo), were provided with standard protocols and instrumentation settings to analyze unknown samples and internal standards in a digested plasma matrix to quantify 51 peptides from 39 human proteins using a multiplexed SRM assay. The interlaboratory coefficient of variation (CV) was less than 10% for 25 of 39 peptides quantified (12 peptides were not quantified based upon hydrophobicity) and exhibited CVs less than 20% for the remaining peptides. In this report, we demonstrate that previously developed research platforms for SRM assays can be improved and optimized for deployment in clinical research environments.

Overexpression of FOXG1 contributes to TGF-beta resistance through inhibition of p21WAF1/CIP1 expression in ovarian cancer.

BACKGROUND: Loss of growth inhibitory response to transforming growth factor-beta (TGF-beta) is a common feature of epithelial cancers. Recent studies have reported that genetic lesions and overexpression of oncoproteins in TGF-beta/Smads signalling cascade contribute to the TGF-beta resistance. Here, we showed that the overexpressed FOXG1 was involved in attenuating the anti-proliferative control of TGF-beta/Smads signalling in ovarian cancer. METHODS: FOXG1 and p21(WAF1/CIP1) expressions were evaluated by real-time quantitative reverse-transcription polymerase chain reaction (RT-PCR), western blot and immunohistochemical analyses. The effect of FOXG1 on p21(WAF1/CIP1) transcriptional activity was examined by luciferase reporter assays. Cell lines stably expressing or short hairpin RNA interference-mediated knockdown FOXG1 were established for studying the gain-or-loss functional effects of FOXG1. XTT cell proliferation assay was used to measure cell growth of ovarian cancer cells. RESULTS: Quantitative RT-PCR and western blot analyses showed that FOXG1 was upregulated and inversely associated with the expression levels of p21(WAF1/CIP1) in ovarian cancer. The overexpression of FOXG1 was significantly correlated with high-grade ovarian cancer (P=0.025). Immunohistochemical analysis on ovarian cancer tissue array was further evidenced that FOXG1 was highly expressed and significantly correlated with high-grade ovarian cancer (P=0.048). Functionally, enforced expression of FOXG1 selectively blocked the TGF-beta-induced p21(WAF1/CIP1) expressions and increased cell proliferation in ovarian cancer cells. Conversely, FOXG1 knockdown resulted in a 20-26% decrease in cell proliferation together with 16-33% increase in p21(WAF1/CIP1) expression. Notably, FOXG1 was able to inhibit the p21(WAF1/CIP1) promoter activity in a p53-independent manner by transient reporter assays. CONCLUSION: Our results suggest that FOXG1 acts as an oncoprotein inhibiting TGF-beta-mediated anti-proliferative responses in ovarian cancer cells through suppressing p21(WAF1/CIP1) transcription.

Molecular cloning and mRNA distribution of pituitary adenylate cyclase-activating polypeptide (PACAP)/PACAP-related peptide in the lungfish.

In this article, we report the isolation of a full-length cDNA clone encoding pituitary adenylate cyclase-activating polypeptide (PACAP)/PACAP-related peptide (PRP) from lungfish Protopterus dolloi. When comparing the deduced amino acid sequences, the lungfish PACAP was found to be highly conserved with other vertebrates; however, the PRP shares only lower levels of sequence identity with known PRP sequences. Consistently in phylogenetic analysis, the lungfish PRP, similar to sturgeon PRP, fails to cluster with other PRPs. In addition to the full-length clone, another cDNA encoding a short precursor that lacks the first 32 amino acids of the PRP was also isolated. Interestingly, similar isoforms were also identified in several nonmammalian vertebrates, and it was suggested that exon skipping of PRP/PACAP transcripts was a mechanism that regulated the expression ratio of PACAP to PRP in nonmammalian vertebrates. By real-time PCR, both long and short PRP/PACAP transcripts were found almost exclusively in the brain, and the short isoform is the more abundant transcript (3.7 times more), indicating that PACAP is the major product produced in lungfish brain. The expression patterns of lungfish and previously studied frog PRP/PACAP suggest that the PRP/PACAP gene in the tetrapod lineage may first express in the central nervous system; in the process of evolution, the functions of these peptides diversified and were later found in other tissues.

Over-expression of FOXM1 transcription factor is associated with cervical cancer progression and pathogenesis.

The Forkhead Box M1 (FOXM1) transcription factor plays a crucial role in regulating expression of cell cycle genes which are essentially involved in cell proliferation, differentiation and transformation. Recent studies have reported that aberrant expression of FOXM1 in a variety of human cancers is associated with their aggressive behaviour. However, the functional significance of FOXM1 in human cervical cancer is not known. We have shown that FOXM1 was significantly over-expressed in cervical squamous cell carcinoma (SCC) compared to normal cervical epithelium immunohistochemically (p < 0.001). In addition, intratumoural FOXM1 positivity was increased in cervical intraepithelial neoplasia (CIN) and carcinoma, compared with that in normal epithelium, indicating that FOXM1 is involved in tumour progression. Indeed, this is supported by clinicopathological analysis that the over-expression of FOXM1 was significantly associated with tumour late stage (p = 0.012) and cell proliferation marker, Ki67 (p < 0.001). Functionally, enforced expression of FOXM1c in FOXM1-deficient cervical cancer cells (C33A) remarkably enhanced cell proliferation and anchorage-independent growth ability. Conversely, depletion of FOXM1 by RNA interference in FOXM1-over-expressing cervical cancer cells (SiHa) caused significant inhibition on cell proliferation and anchorage-independent growth ability on soft agar. This inhibitory phenomenon was associated with the reduced expressions of cyclin B1, cyclinD1 and cdc25B but increased expression of p27(Kip1) and p21(Cip1). Our findings suggest a role for FOXM1 in the development and pathogenesis of human cervical SCC.

Expression and clinicopathological significance of oestrogen-responsive ezrin-radixin-moesin-binding phosphoprotein 50 in breast cancer.

AIMS: Ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50) is a post synaptic density-95/Disk-large/ZO-1 homologous domain-containing protein that is involved in the linkage of integral membrane proteins to the cytoskeleton and plays an important role in cell signalling. To gain insights into its biological relevance, this study examined expression of EBP50 in two cohorts of breast carcinoma. METHODS AND RESULTS: Forty-nine breast carcinoma tissue specimens were first examined by both immunohistochemistry and RNA in situ hybridization. EBP50 expression was correlated with various clinicopathological variables. The relative abundance of EBP50 mRNA in breast carcinomas and their corresponding normal tissue was compared using reverse transcriptase-polymerase chain reaction (RT-PCR). EBP50 immunoreactivity was then further independently validated in 120 breast carcinomas on tissue microarrays. EBP50 immunoreactivity was observed in morphologically normal and cancerous epithelial cells contrasting with the adjacent immunonegative stromal cells. An elevated cytoplasmic accumulation of EBP50 protein was readily detected in 73.5-80% of breast carcinomas. EBP50 immunoreactivity was significantly associated with tumour stage, lymph node and oestrogen receptor status. These immunohistochemical observations were further validated using RNA in situ hybridization and RT-PCR. EBP50 immunoreactivity was significantly correlated with the mRNA expression level. CONCLUSION: Oestrogen-responsive EBP50 may play an important role in tumour progression and might be a potential marker of invasiveness for breast cancer.

Targeted gene transfer to Schlemm's canal by retroperfusion.

The goal of the present study was to specifically modify protein expression in the resistance-generating region of the conventional outflow pathway, namely the inner wall of Schlemm's canal (SC) and the juxtacanalicular region of the trabecular meshwork, in perfused human anterior segments. Anterior segments from human cadaveric eyes were prepared for organ culture using standard techniques and were perfused at constant flow while recording pressure. After reaching a stable outflow facility within physiological limits, forward perfusion was stopped and a fluid-tight fence encircling the limbus was installed and filled with media containing an adenovirus encoding the lacZ reporter gene (either 2 x 10(6) or 6 x 10(6)PFU/ml). With the limbus submerged, pressure inside the chamber was lowered to -1 mmHg to facilitate reverse perfusion of virus into SC ("retroperfusion"). After 30-60 min at zero pressure (with some mixing), forward perfusion was restarted and continued for 5-7 days, after which anterior segments were fixed and processed for visualization of lacZ activity. Retroperfusion of nine anterior segments with adenovirus encoding a reporter gene did not appreciably alter baseline outflow facility (0.27+/-0.05 versus 0.29+/-0.08 microl/min per mmHg post-retroperfusion). Gross examination of outflow tissues showed focal distribution of lacZ activity around the circumference of SC, presumably near collector channels. In segments that were sequentially tilted during retroperfusion, the distribution of lacZ activity appeared more uniform. Sagittal histological sections showed lacZ activity in all portions of the conventional drainage tract, particularly cells in the resistance-generating region. Taken together, the results demonstrate that candidate protein expression by cells in the resistance-generating region of the conventional drainage pathway can be specifically modified by retroperfusion of adenovirus and examined for effects on outflow facility.

Proteomic analysis of dunning prostate cancer cell lines with variable metastatic potential using SELDI-TOF.

BACKGROUND: Surface enhanced laser desorption and ionization-time-of-flight (SELDI-TOF) is an evolving proteomic technology for improving biomarker discovery that allows for rapid and sensitive analysis of complex protein mixtures generated from body fluids, cells, and/or tissues. SELDI--based profiling identifies unique, differentially expressed proteins relating to specific cancer-related disease states. We utilized SELDI-TOF following pre-processing with molecular separation and chemical fractionation of cell membrane extracts from three Dunning rat prostate cancer cell lines of varying metastatic potential to search novel proteins that are differentially expressed. METHODS: Dunning rat cell sublines of variable (%) metastatic potential; G (0%), AT-1 (20%), and Mat-Ly-Lu (100%) were cultured in two different laboratories. Cell lysis was performed in a homogenation buffer (320 mM sucrose/50 mM Tris/0.5 mM PSMF) using Dounce homogenation. After centrifugation, the membrane pellet was washed 2x and then solublized in 2% CHAPS/8 M urea. This sample was further processed using positive pressure molecular ultrafiltration at 30 kDa or precipitation with 50% ammonium sulfate. Next, each sample was applied to an IMAC3-Ni ProteinChip (Ciphergen Biosystems, Freemont, CA) and analyzed using Ciphergen's Protein Biology System with protein peak analysis software. RESULTS: SELDI-TOF analysis differentiated the three Dunning rat cell sublines based upon protein concentration normalized profiles between 5,000 and 20,000 Da. The preparations from the three cells lines showed clear differences when the extracts from the metastatic sublines (AT-1 and MLL) were compared to the benign subline (G) for proteins with molecular weights of 9 kDa (decrease), 12 kDa (significant decrease), 14 kDa (decrease), and 17 kDa (significant gain). After pre-processing extracts with ammonium sulfate and molecular ultrafiltration, the molecular profile changes from one subline to the next became more apparent. Our results were reproducible using multiple runs including from Dunning cells cultured in a separate laboratory, and using different lots of SELDI ProteinChips. CONCLUSIONS: The application of SELDI-TOF to a series of Dunning rat prostate cancer cell lines illustrated apparent changes in protein profiles among the three cell lines with known differences in metastatic biologic activity. SELDI-TOF identified four reproducible changes in protein expression in the AT1 and MLL metastatic cell sublines. Three of the expression changes were manifested as decreases, but one protein (17 kDa) was over-expressed in the AT1 and MLL cell lines. Emphasis will be placed on the isolation, purification, and characterization of the 17 kDa over-expressed protein and its potential role in PCa metastasis.

Estimating the total exposure to air pollutants for different population age groups in Hong Kong.

Given that Hong Kong is one of the most densely populated cities in the world, the exposure of the Hong Kong people is one of the interesting research areas. In this study, an indirect approach was used to estimate the exposure to nitrogen dioxide (NO2), respiratory dust (PM10) and carbon monoxide (CO) pollutants experienced by different age groups of people in Hong Kong. The average concentrations of the 20 major microenvironments obtained from our measurement survey data, together with the people activity pattern data obtained from 7-day recall questionnaires, were used to predict frequency distributions to exposure assessment. Our results showed that Hong Kong people spent more than 86% of their time indoors. Homes were shown to be the one of the major exposure sites to NO2, CO and PM10 for all age groups. Our results also indicate that the 24-h NO2 exposure for individuals, irrespective of age, spending more than 2 h in commuting daily, was observed to be exceeding the 24-h NO2 exposure standards. This study was one of the pioneering studies with valuable contribution for modeling the estimates of exposures to NO2, PM10 and CO of different age groups in Hong Kong.

Plasma selenium level before diagnosis and the risk of prostate cancer development.

PURPOSE: Epidemiological studies and a randomized intervention trial suggest that the risk of prostate cancer may be reduced by selenium intake. We investigated whether plasma selenium level before diagnosis correlated with the risk of later developing prostate cancer. MATERIALS AND METHODS: A case control study was performed on men from the Baltimore Longitudinal Study of Aging registry, including 52 with known prostate cancer and 96 age matched controls with no detectable prostatic disease. Plasma selenium was measured at an average time plus or minus standard deviation of 3.83 +/- 1.85 years before the diagnosis of prostate cancer by graphite furnace atomic absorption spectrophotometry. Adjusted odds ratio and 95% confidence interval were computed with logistic regression. RESULTS: After correcting for years before diagnosis, body mass index, and smoking and alcohol use history, higher selenium was associated with a lower risk of prostate cancer. Compared with the lowest quartile of selenium (range 8.2 to 10.7 microg./dl.), the odds ratios of the second (10.8 to 11.8), third (11.9 to 13.2) and fourth (13.3 to 18.2) quartiles were 0.15 (95% confidence interval 0.05 to 0.50), 0.21 (0.07 to 0.68) and 0.24 (0.08 to 0.77, respectively, p =0.01). Furthermore, plasma selenium decreased significantly with patient age (p <0.001). CONCLUSIONS: Low plasma selenium is associated with a 4 to 5-fold increased risk of prostate cancer. These results support the hypothesis that supplemental selenium may reduce the risk of prostate cancer. Because plasma selenium decreases with patient age, supplementation may be particularly beneficial to older men.

Immunosensors--principles and applications to clinical chemistry.

INTRODUCTION: Immunosensors are affinity ligand-based biosensor solid-state devices in which the immunochemical reaction is coupled to a transducer. The fundamental basis of all immunosensors is the specificity of the molecular recognition of antigens by antibodies to form a stable complex. This is similar to the immunoassay methodology. Immunosensors can be categorized based on the detection principle applied. The main developments are electrochemical, optical, and microgravimetric immunosensors. In contrast to immunoassay, modern transducer technology enables the label-free detection and quantification of the immune complex. METHODS: The analysis of trace substances in environmental science, pharmaceutical and food industries is a challenge since many of these applications demand a continuous monitoring mode. The use of immunosensors in these applications is most appropriate. Similarly, a series of clinical problems may be solved by continuous monitoring of certain analytes. CONCLUSIONS: Clinical chemists should take advantage of immunosensors in clinical diagnostics. There are many recent developments in the immunosensor field which have potential impacts. The future role of this technique in intralaboratory, as well as bedside testing, will become even more important as the clinical laboratory is faced with increasing pressure to contain costs.

Cardiac troponin T and C-reactive protein as markers of acute cardiac allograft rejection.

Due to myocyte damage and an associated inflammatory response, it is possible that cardiac troponin T and C-reactive protein (CRP) concentrations may correlate with the histologic grade of rejection in endomyocardial biopsy samples obtained from patients who have received a heart transplant. In this study, 704 blood samples were obtained from 145 different heart transplant recipients just prior to endomyocardial biopsy. Plasma specimens were assayed for troponin T and CRP concentration and the results compared with the assigned International Society of Heart and Lung Transplantation (ISHLT) histologic grade. Rejection was defined as an ISHLT grade of 3A or higher. The negative predictive values were near 80% in all cases, and a statistically significant increase in median troponin T concentration was observed across ISHLT grades. After the first month posttransplantation, the specificity of the troponin T test (cutoff 0.1 ng/ml) was 95% and increased to 98% when false positives seen in renal disease patients were excluded. Both tests demonstrated poor sensitivity and positive predictive value for rejection. Neither CRP nor troponin T had sufficient sensitivity to serve as an alternative to endomyocardial biopsy in the diagnosis of acute cardiac allograft rejection. However, the troponin T test had a high specificity, especially when patients with renal insufficiency were excluded, and could serve as an adjunct test in this setting. When combined with a normal serum creatinine, a troponin T > or =0.1 ng/ml prior to endomyocardial biopsy correlated with graft rejection in almost all cases, making biopsy unnecessary.

Low levels of prostate-specific antigen predict long-term risk of prostate cancer: results from the Baltimore Longitudinal Study of Aging.

OBJECTIVES: To evaluate the relationship between low prostate-specific antigen (PSA) levels that are considered normal and the long-term risk of prostate cancer. METHODS: The relative risk of, and cumulative probability of freedom from, prostate cancer by PSA level and age decade was evaluated in male participants of a longitudinal aging study, the Baltimore Longitudinal Study of Aging (National Institute on Aging). The relative risk was estimated from a Cox proportional hazards regression model for men aged 40 to 49.9 (n = 351) and 50 to 59.9 (n = 445). The disease-free probability was determined by Kaplan-Meier survival analysis. RESULTS: The relative risk of prostate cancer for men aged 40 to 49.9 was 3.75 (range 1.6 to 8.6) when the PSA level was at or greater than the median (0.60 ng/mL) compared with men with PSA levels less than the median. This risk was similar for men aged 50 to 59.9 when comparing those with PSA levels greater than and less than the median (0.71 ng/mL). At 25 years, the cumulative probability of freedom from prostate cancer for men aged 40 to 49.9 was 89.6% (range 81% to 97%) and 71.6% (range 60% to 83%) when the PSA level was less than and greater than the median, respectively. The 25-year disease-free probability for men aged 50 to 59.9 was 83.6% (range 76% to 91%) and 58.9% (range 48% to 70%) when the PSA level was less than and greater than the median, respectively. CONCLUSIONS: The association between the baseline serum PSA level and the subsequent risk of prostate cancer suggests that the biologic events that predispose to prostate cancer begin early in middle age. Men who have baseline PSA levels that are "normal" but reflect a higher risk of prostate cancer may be the most appropriate candidates for future prevention trials. Those men with the lowest risk of prostate cancer on the basis of the baseline PSA measurements are unlikely to benefit from frequent PSA surveillance in an effort to detect prostate cancer early.

Werner syndrome protein is regulated and phosphorylated by DNA-dependent protein kinase.

DNA double-strand breaks (DSBs) are a highly mutagenic and potentially lethal damage that occurs in all organisms. Mammalian cells repair DSBs by homologous recombination and non-homologous end joining, the latter requiring DNA-dependent protein kinase (DNA-PK). Werner syndrome is a disorder characterized by genomic instability, aging pathologies and defective WRN, a RecQ-like helicase with exonuclease activity. We show that WRN interacts directly with the catalytic subunit of DNA-PK (DNA-PK(CS)), which inhibits both the helicase and exonuclease activities of WRN. In addition we show that WRN forms a stable complex on DNA with DNA-PK(CS) and the DNA binding subunit Ku. This assembly reverses WRN enzymatic inhibition. Finally, we show that WRN is phosphorylated in vitro by DNA-PK and requires DNA-PK for phosphorylation in vivo, and that cells deficient in WRN are mildly sensitive to ionizing radiation. These data suggest that DNA-PK and WRN may function together in DNA metabolism and implicate WRN function in non-homologous end joining.

Cationic ferritin changes outflow facility in human eyes whereas anionic ferritin does not.

PURPOSE: To determine the effect of charged moieties within the outflow pathway on aqueous outflow facility in human eyes. METHODS: After baseline facility measurement in human eye bank eyes (n = 10 pairs), one eye of each pair received anterior chamber exchange and continued perfusion with medium containing 10 mg/ml cationic ferritin. Contralateral eyes were treated in a similar manner with anionic ferritin (10.0 or 102 mg/ml). Eyes were fixed by anterior chamber exchange and perfusion with universal fixative at 8 mm Hg (corresponding to a physiologic pressure of 15 mm Hg in vivo) and examined by transmission electron microscopy. In a second series of human eyes (n = 8 pairs), facility was measured before and after anterior chamber exchange, with a solution containing 0.1 U/ml neuraminidase. RESULTS: Perfusion of eyes with anionic ferritin at either 10.0 or 102 mg/ml caused a negligible 2% increase in facility, whereas cationic ferritin perfusion reduced facility by 66% (P < 0.00001). Perfusion with fixative reduced facility by approximately 60% in both cationic and anionic ferritin-perfused eyes, relative to facilities after perfusion with ferritin. Transmission electron microscopy showed that the distribution of ferritin was segmentally variable. Cationic ferritin consistently labeled the luminal surface of the inner wall of Schlemm's canal, and variably labeled the juxtacanalicular connective tissue (JCT) and trabecular beam surfaces. Anionic ferritin was more prominent in the JCT and intertrabecular spaces and less so on the luminal surface of Schlemm's canal. By scanning electron microscopy, cationic ferritin was seen to accumulate at intercellular margins of the inner wall. Neuraminidase perfusion had no significant effect on outflow facility. CONCLUSIONS: Cationic ferritin reduces outflow facility, presumably by binding to negatively charged sites in the outflow pathway. A possible mechanism is partial or complete blockage of intercellular clefts in the inner wall of Schlemm's canal by the ferritin that accumulates on the luminal surface of the inner wall. Although they are possible targets for ferritin binding, sialyl residues themselves seem to have little direct effect on outflow facility. Our data indicate that positively charged molecules, especially if they can interact with inner wall pores, have the potential to markedly alter outflow facility.

Concentration of enzymatically active prostate-specific antigen (PSA) in the extracellular fluid of primary human prostate cancers and human prostate cancer xenograft models.

BACKGROUND: Prostate-specific antigen (PSA) targeted prodrugs are under development in our laboratory. Concentrations of total PSA and enzymatically active PSA produced by various human prostate cancer xenograft models have not been well characterized. METHODS: The concentration of PSA secreted into the extracellular fluid (ECF) in normal human prostate tissue, primary prostate cancers obtained directly from patients, and serially passageable human prostate cancer xenografts (PC-82, LNCaP, LAPC-4) were determined using Tandem assays. Percent enzymatically active PSA in the ECF and in conditioned media was also determined using a previously validated assay employing a monoclonal antibody to the PSA catalytic site. In addition, the concentration and activity of PSA within sera from men with and without prostate cancer, as well as from tumor-bearing animals, was likewise assayed. RESULTS: Normal human prostate tissue and primary human prostate cancers have high concentrations of PSA in the ECF (i.e., 1600-2100 nM). The majority of this PSA is enzymatically active (i.e., 80-90%). Human PC-82 prostate cancer xenografts also have high concentrations of PSA in the ECF (624 +/- 360 nM), and the majority of this PSA is also enzymatically active (i.e., 66 +/- 4%). In contrast, much lower concentrations of PSA are found in the ECF from LNCaP (45 +/- 9 nM) and LAPC-4 (7.3 +/- 0.6 nM). Only a small portion of the total PSA isolated from DHT-containing, serum-free, conditioned media from these cell lines is enzymatically active (i.e., approximately 18%). While PSA was detected in all serum samples regardless of the type of host, no enzymatically active PSA was detected in any of these serum samples. CONCLUSIONS: Prostate cancers obtained directly from patients produce and secrete large amounts of PSA, the majority of which is highly enzymatically active. In contrast, while PSA was detected in the sera, none of this PSA was enzymatically active. This is also the case for the human PC-82 prostate cancer xenografts. In contrast, LNCaP and LAPC-4 human prostate cancer xenograft models secrete approximately 70-300-fold less PSA in the ECF than prostate cancers from patients and the majority of this PSA is enzymatically inactive. Also, the serum from these animals had detectable PSA, but none of this PSA was enzymatically active. Thus, these latter two prostate cancer models define the least and the PC-82, the most, optimized xenograft model for screening PSA targeted prodrugs.