Forkhead box M1 (FOXM1) transcription factor is a key oncogenic driver of aggressive human meningioma progression.

AIMS: Aggressive meningioma remains incurable with neither chemo- nor targeted therapies proven effective, largely due to unidentified genetic alterations and/or aberrant oncogenic pathways driving the disease progression. In this study, we examined the expression and function of Forkhead box M1 (FOXM1) transcription factor during meningioma progression. METHODS: Human meningioma samples (n = 101) were collected, followed by Western blotting, quantitative PCR, immunohistochemical and progression-free survival (PFS) analyses. For in vitro assays, FOXM1 was overexpressed or knocked-down in benign (SF4433 and SF4068) or malignant (SF3061 and IOMM-Lee) human meningioma cell lines respectively. For in vivo studies, siomycin A (a FOXM1 inhibitor)-pretreated or control IOMM-Lee cells were implanted subcutaneously in nude mice. RESULTS: FOXM1 expression was increased in higher grades of meningioma and correlated with the mitotic index in the tumour tissue. Moreover, FOXM1 was increased in recurrent meningioma compared with the matched primary lesions. The patients who had higher FOXM1 expression had shorter PFS. In the subsequent in vitro assays, knockdown of FOXM1 in malignant meningioma cell lines resulted in decreased tumour cell proliferation, angiogenesis and invasion, potentially via regulation of ß-catenin, cyclin D1, p21, interleukin-8, vascular endothelial growth factor-A, PLAU, and epithelial-to-mesenchymal transition-related genes, whereas overexpression of FOXM1 in benign meningioma cell lines had the opposite effects. Last, suppression of FOXM1 using a pharmacological inhibitor, siomycin A, decreased tumour growth in an in vivo mouse model. CONCLUSIONS: Our data demonstrate that FOXM1 is a key transcription factor regulating oncogenic signalling pathways in meningioma progression, and a promising therapeutic target for aggressive meningioma.

Detection of viable but nonculturable Vibrio parahaemolyticus induced by prolonged cold-starvation using propidium monoazide real-time polymerase chain reaction.

Viable but nonculturable (VBNC) Vibrio parahaemolyticus cannot be detected by the standard cultivation-based methods. In this study, commonly used viability assessment methods were evaluated for the detection of V. parahaemolyticus in a VBNC state. Vibrio parahaemolyticus cells exposed to nutrient deficiency at cold temperature were used for epifluorescence microscopy with SYTO9 and propidium iodide (PI) staining and real-time polymerase chain reaction (qPCR) with propidium monoazide (PMA), and its resuscitative ability was determined by a temperature upshift in freshly prepared artificial sea water (ASW; pH 7) fluids. Viable cells with intact membranes always exceeded 5·0 log CFU per ml in ASW microcosms at 4°C. After 80 days, cycle thresholds for V. parahaemolyticus ATCC 27969 were 16·15-16·69. During cold-starvation, PMA qPCR selectively excluded DNAs from heat-killed cells. However, there may be some penetration of PMA into undamaged cells that persisted in ASW for 150 days, as evidenced by their ability to resuscitate from a VBNC state after a temperature upshift (25°C); V. parahaemolyticus ATCC 33844 and V. parahaemolyticus ATCC 27969 were successfully reactivated from a VBNC state in ASW microcosms containing <5% NaCl, following enrichment in ASW medium (pH 7). SIGNIFICANCE AND IMPACT OF THE STUDY: Few studies have evaluated the characteristics of and detection methods for viable but nonculturable (VBNC) Vibrio parahaemolyticus induced by cold-starvation. Currently, VBNC cells are routinely detected by SYTO9 and propidium iodide double staining. However, viable cell counts might be overestimated by this approach, suggesting that the fluorescence dyes may be ineffective for accurately determining the viability of bacterial cells. We demonstrated that quantitative real-time polymerase chain reaction with propidium monoazide, which selectively permeates damaged cell membranes, can be used to obtain viable cell counts of V. parahaemolyticus after its evolution to a VBNC state under cold-starvation conditions.

Neuropilin-2 gene expression correlates with malignant progression in cutaneous melanoma.

BACKGROUND: It is currently not possible to predict the metastatic potential of early-stage melanoma lesions by histological examination alone; however, a significant number of thin melanomas will progress over time to advanced disease. Molecular biomarkers that could identify patients with melanoma at high risk at the time of original diagnosis would contribute significantly to improved patient outcomes and increased survival. Neuropilin-2 (NRP2), a cell surface receptor involved in tumour-associated angiogenesis and lymphangiogenesis, has recently been shown to be expressed in melanoma. OBJECTIVES: To evaluate the potential value of NRP2 gene transcript levels as biomarkers for malignant melanoma progression. METHODS: We measured NRP2 gene expression in a panel of formalin-fixed paraffin-embedded tissue specimens consisting of naevi, primary melanomas and metastatic melanomas using quantitative reverse transcriptase-polymerase chain reaction technique. RESULTS: NRP2 levels are clearly segregated among the groups of naevi, primary and metastatic melanoma samples with a statistical trend towards increasing NRP2 gene expression correlating with disease progression. Logistic regression analysis reveals that the probability of malignant progression increases with elevated levels of NRP2 (odds ratio of 2·60 with confidence interval 1·29-5·21). Within the group of primary melanomas, there is a positive correlation (r = 0·823) between NRP2 expression and Breslow depth. This correlation was validated in an independent sample set of patients with melanoma. CONCLUSIONS: This preliminary study strongly supports the significance of NRP2 as a useful biomarker for malignant progression of melanoma, which may be useful for early identification of patients with melanoma at high risk.

Optimizing facility location, sizing, and growth time for a cultivated resource: A case study in coral aquaculture.

Production of cultivated resources require additional planning that takes growth time into account. We formulate a mathematical programming model to determine the optimal location and sizing of growth facilities, impacted by resource survival rate as a function of its growth time. Our method informs strategic decisions regarding the number, location, and sizing of facilities, as well as operational decisions of optimal growth time for a cultivated resource in a facility to minimize total costs. We solve this facility location and sizing problem in the context of coral aquaculture for large-scale reef restoration using a two-stage algorithm and a linear mixed-integer solver. We assess growth time in a facility in terms of its impact on survival (post-deployment) considering growth quantity requirements and growth facility production constraints. We explore the sensitivity of optimal facility number, location, and sizing to changes in the geographic distribution of demand and cost parameters computationally. Results show that the relationship between growth time and survival is critical to optimizing operational decisions for grown resources. These results inform the value of data certainty to optimize the logistics of coral aquaculture production.

Effects of subinhibitory concentrations of chlorhexidine and mupirocin on biofilm formation in clinical meticillin-resistant Staphylococcus aureus.

BACKGROUND: The effects of subinhibitory concentrations (sub-MICs) of antibacterial agents on the biofilm-forming ability of Staphylococcus aureus require further study. AIM: To investigate the effects of sub-MICs of chlorhexidine and mupirocin on biofilm formation in clinical meticillin-resistant Staphylococcus aureus (MRSA) isolates. METHODS: MRSA isolates were collected from patients with bloodstream infections at a tertiary care hospital. The basal level of biofilm formation and biofilm induction by sub-MICs of chlorhexidine and mupirocin were evaluated by measuring biofilm mass stained with Crystal Violet. FINDINGS: Of the 112 MRSA isolates tested, 63 (56.3%) and 44 (39.3%) belonged to sequence type (ST)5 and ST72 lineages, respectively, which are the predominant healthcare- and community-associated clones in South Korea. ST5 isolates were more likely to have chlorhexidine MIC ≥4 (73.0% vs 29.5%), resistance to mupirocin (23.8% vs 0%), agr dysfunction (73.0% vs 9.1%), and qacA/B gene (58.7% vs 2.3%) compared to ST72 isolates. The basal level of biofilm formation ability was frequently stronger in ST72 isolates compared to ST5 isolates (77.3% vs 12.7%). Sub-MICs of chlorhexidine and mupirocin promoted biofilm formation in 56.3% and 53.6%, respectively, of all isolates. Biofilm induction was more prevalent in ST5 isolates (85.7% for chlorhexidine, 69.8% for mupirocin) than in ST72 isolates (15.9% for chlorhexidine, 27.3% for mupirocin). CONCLUSION: Sub-MICs of chlorhexidine and mupirocin promoted biofilm formation in half of the clinical MRSA isolates. Our results suggest that ST5 MRSA biofilm can be induced together with some other bacterial virulent factors following exposure to chlorhexidine, which might confer a survival advantage to this clone in the healthcare environment.

Extensive severe fever with thrombocytopenia syndrome virus contamination in surrounding environment in patient rooms.

OBJECTIVES: Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne disease in Korea and China. Although there is previous evidence of person-to-person transmission via direct contact with body fluids, the role of environmental contamination by SFTS virus (SFTSV) in healthcare settings has not been established. We therefore investigated the contamination of the healthcare environment by SFTSV. METHODS: We investigated the possible contamination of hospital air and surfaces with SFTSV transmission by collecting air and swabbing environmental surface samples in two hospitals treating six SFTS patients between March and September 2017. The samples were tested using real-time RT-PCR for SFTS M and S segments. RESULTS: Of the six SFTS patients, four received mechanical ventilation and three died. Five rooms were occupied by those using mechanical ventilation or total plasma exchange therapy in isolation rooms without negative pressure and one room was occupied by a patient bedridden due to SFTS. SFTSV was detected in 14 (21%) of 67 swab samples. Five of 24 swab samples were obtained from fomites including stethoscopes, and 9 of 43 were obtained from fixed structures including doorknobs and bed guardrails. Some samples from fixed structures such as television monitors and sink tables were obtained in areas remote from the patients. SFTSV RNA was not detected in five air samples from three patients' rooms. CONCLUSIONS: Our data suggest that SFTSV contamination was extensive in surrounding environments in SFTS patients' rooms. Therefore, more strict isolation methods and disinfecting procedures should be considered when managing SFTS patients.

Promoter CpG hypermethylation and downregulation of XAF1 expression in human urogenital malignancies: implication for attenuated p53 response to apoptotic stresses.

XIAP-associated factor 1 (XAF1) is a new candidate tumor suppressor, which has been known to exert proapoptotic effects by interfering with the caspase-inhibiting activity of XIAP. To explore the XAF1's candidacy for a suppressor in urogenital tumorigenesis, we investigated the XAF1 status in a series of cancer cell lines and primary tumors derived from the bladder, kidney and prostate. Expression of XAF1 transcript was undetectable or extremely low in 60% (3/5) of bladder, 66% (10/15) of kidney, and 100% (3/3) prostate cancer cell lines. Abnormal reduction of XAF1 was also found in 33% (18/55) of primary bladder and 40% (8/20) of primary kidney tumors, and showed a correlation with advanced stage and high grade of bladder tumor. Hypermethylation at 14 CpG sites in the 5' proximal region of the XAF1 promoter was highly prevalent in cancers versus adjacent normal or benign tissues and tightly associated with reduced gene expression. XAF1 expression enhanced the apoptotic response of tumor cells to chemotherapeutic agents, such as etoposide or 5-FU. While XAF1 expression did not influence the subcellular distribution or expression of XIAP, it elevated the protein stability of p53 and its target gene expression. Moreover, the apoptosis-sensitizing and growth suppression function of XAF1 was markedly impeded by blockade of p53 function. Collectively, our study demonstrates that epigenetic alteration of XAF1 is frequent in human urogenital cancers and may contribute to the malignant progression of tumors by rendering tumor cells a survival advantage partially through the attenuated p53 response to apoptotic stresses.

Structure and dynamic properties of a glucocorticoid receptor-induced chromatin transition.

Activation of the mouse mammary tumor virus (MMTV) promoter by the glucocorticoid receptor (GR) is associated with a chromatin structural transition in the B nucleosome region of the viral long terminal repeat (LTR). Recent evidence indicates that this transition extends upstream of the B nucleosome, encompassing a region larger than a single nucleosome (G. Fragoso, W. D. Pennie, S. John, and G. L. Hager, Mol. Cell. Biol. 18:3633-3644). We have reconstituted MMTV LTR DNA into a polynucleosome array using Drosophila embryo extracts. We show binding of purified GR to specific GR elements within a large, multinucleosome array and describe a GR-induced nucleoprotein transition that is dependent on ATP and a HeLa nuclear extract. Previously uncharacterized GR binding sites in the upstream C nucleosome region are involved in the extended region of chromatin remodeling. We also show that GR-dependent chromatin remodeling is a multistep process; in the absence of ATP, GR binds to multiple sites on the chromatin array and prevents restriction enzyme access to recognition sites. Upon addition of ATP, GR induces remodeling and a large increase in access to enzymes sites within the transition region. These findings suggest a dynamic model in which GR first binds to chromatin after ligand activation, recruits a remodeling activity, and is then lost from the template. This model is consistent with the recent description of a "hit-and-run" mechanism for GR action in living cells (J. G. McNally, W. G. Müller, D. Walker, and G. L. Hager, Science 287:1262-1264, 2000).

A novel histone deacetylase inhibitor identified by high-throughput transcriptional screening of a compound library.

Libraries of compounds are increasingly becoming commercially available for the use of individual academic laboratories. A high-throughput system based on a stably integrated transcriptional reporter was used to screen a library of random compounds to identify agents that conferred robust augmentation of a signal transduction pathway. A novel histone deacetylase (HDAC) inhibitor, termed scriptaid, conferred the greatest effect, a 12- to 18-fold augmentation. This facilitation of transcriptional events was generally applicable to exogenous gene constructs, including viral and cellular promoters, different cell lines and reporter genes, and stably integrated and transiently introduced sequences. Scriptaid did not interfere with a further induction provided by stimulation of the cognate signal transduction pathway (transforming growth factor beta/Smad4), which implied the functional independence of ligand-stimulated transcriptional activation and histone acetylation states in this system. Additional insights into this and other signal transduction systems are likely to be afforded through the application of compound screening technologies.

Invasion-specific genes in malignancy: serial analysis of gene expression comparisons of primary and passaged cancers.

The invasive growth of malignant cells induces an admixture of host reactions including desmoplasia, angiogenesis, and immune reactions Pancreatic cancer has a prominent and characteristic host reaction at the site of primary invasion. To obtain new insights into the process of tumor invasion, we studied global patterns of gene expression using serial analysis of gene expression in pancreatic cancer, with extension to other tumor types. Here we report a cluster of invasion-specific genes in pancreatic and other cancers. This cluster contains genes that derive from distinct components of the host reaction, including some that may be useful as diagnostic markers and therapeutic targets.

Frequent epigenetic inactivation of RASSF1A by aberrant promoter hypermethylation in human gastric adenocarcinoma.

Methylation associated inactivation of RASSF1, a putative tumor suppressor identified at 3p21.3, has been frequently observed in several human malignancies, including lung and breast cancers. To explore the penetrance of RASSF1 in gastric carcinogenesis, we performed expression and mutation analyses of 3 isotypes of RASSF1 (A, B, and C) in 150 gastric specimens, including 15 carcinoma cell lines. RASSF1A and RASSF1B transcripts were not expressed in 60% (9 of 15) and 33% (5 of 15) of gastric carcinoma cell lines, respectively, whereas RASSF1C was detectable in all cell lines. Bisulfite DNA sequencing analysis revealed that the CpG island in the RASSF1A promoter is hypermethylated in all RASSF1A-nonexpressing cell lines. In addition, both RASSF1A and RASSF1B were re-expressed by treatment with the demethylating agent 5-aza-2'-deoxycytidine. Among 90 primary gastric adenocarcinomas examined, 41 (46%) and 19 (21%) expressed no or abnormally low levels of RASSF1A and RASSF1B, respectively, and 12 (13%) tumors showed no expression of both isoforms. Loss or abnormal down-regulation of RASSF1A correlated with tumor stage and grade but not with histological types of tumors. Methylation-specific PCR analysis demonstrated that 95% (39 of 41) of RASSF1A-nonexpressing primary tumors are methylated at the CpG sites in the promoter, whereas none of the adjacent noncancerous or normal tissues are methylated. No somatic mutations were detected in RASSF1 transcripts expressed in unmethylated tumors. However, 10 methylated tumors, including 4 cell lines, showed low genomic levels of RASSF1 and expressed no RASSF1A transcripts, suggesting that RASSF1A inactivation might be caused by both epigenetic and genetic mechanisms in a subset of gastric adenocarcinomas. In conclusion, our data indicate that epigenetic transcriptional silencing of RASSF1, especially RASSF1A isoform, is a frequent event in gastric tumorigenesis and might play an important role in the malignant progression of gastric adenocarcinomas.

Discovery of new markers of cancer through serial analysis of gene expression: prostate stem cell antigen is overexpressed in pancreatic adenocarcinoma.

Serial analysis of gene expression (SAGE) can be used to quantify gene expression in human tissues. Comparison of gene expression levels in neoplastic tissues with those seen in nonneoplastic tissues can, in turn, identify novel tumor markers. Such markers are urgently needed for highly lethal cancers like pancreatic adenocarcinoma, which typically presents at an incurable, advanced stage. The results of SAGE analyses of a large number of neoplastic and nonneoplastic tissues are now available online, facilitating the rapid identification of novel tumor markers. We searched an online SAGE database to identify genes preferentially expressed in pancreatic cancers as compared with normal tissues. SAGE libraries derived from pancreatic adenocarcinomas were compared with SAGE libraries derived from nonneoplastic tissues. Three promising tags were identified. Two of these tags corresponded to genes (lipocalin and trefoil factor 2) previously shown to be overexpressed in pancreatic carcinoma, whereas the third tag corresponded to prostate stem cell antigen (PSCA), a recently discovered gene thought to be largely restricted to prostatic basal cells and prostatic adenocarcinomas. PSCA was expressed in four of the six pancreatic cancer SAGE libraries, but not in the libraries derived from normal pancreatic ductal cells. We confirmed the overexpression of the PSCA mRNA transcript in 14 of 19 pancreatic cancer cell lines by reverse transcription-PCR, and using immunohistochemistry, we demonstrated PSCA protein overexpression in 36 of 60 (60%) primary pancreatic adenocarcinomas. In 59 of 60 cases, the adjacent nonneoplastic pancreas did not label for PSCA. PSCA is a novel tumor marker for pancreatic carcinoma that has potential diagnostic and therapeutic implications. These results establish the validity of analyses of SAGE databases to identify novel tumor markers.

Association of negatively-charged phospholipids with low-density lipoprotein (LDL) increases its uptake and the deposition of cholesteryl esters by macrophages.

LDL, the major carrier of cholesterol in blood, is poorly metabolized by macrophages. In contrast, macrophages can recognize and endocytose anionic phospholipids such as phosphatidylserine, phosphatidylglycerol and cardiolipin. Since macrophages can take up large amounts of these phospholipids, experiments were performed to ascertain whether pre-incubation of native LDL with negatively-charged phospholipids would enhance the metabolism of LDL by macrophages. When 125I-LDL was incubated with cardiolipin liposomes for 18 h at 37 degrees C before addition to macrophages, an approx. 40-fold increase of LDL metabolism by these cells was observed. Similar results were found when LDL was pre-incubated with phosphatidylserine or phosphatidylglycerol; however, pre-incubation of LDL with phosphatidylcholine liposomes did not lead to an increase of LDL metabolism. The macrophage uptake of LDL pre-incubated with cardiolipin was reduced to approx. 40% of control values in the presence of dextran sulfate and fucoidin, inhibitors of anionic phospholipid uptake. Cytochalasin D, an inhibitor of phagocytosis, reduced the lysosomal degradation of LDL pre-incubated with cardiolipin to approx. 10% of control values. When the LDL-cardiolipin mixture was chromatographed on agarose gel, two peaks containing LDL were observed in the elution profile: the first peak appeared at the void volume and the second peak was detected just ahead of native LDL. The LDL in both peaks was much more extensively metabolized by macrophages than was native LDL; the LDL in the first peak was metabolized at a rate that was 8 times the second peak. The results demonstrate that negatively-charged phospholipids can form a complex with LDL which facilitates its phagocytosis by macrophages.

Iron-ascorbate-phospholipid mediated modification of low density lipoprotein.

LDL can be oxidized by a variety of agents to form a modified lipoprotein which is capable of being avidly metabolized by macrophages. While previous in vitro studies have focused exclusively on the oxidation of LDL, other lipids found in the atheroma are also subject to oxidation and its lipoperoxide byproducts may contribute to the process of LDL modification. To examine the relationship between the oxidation of phospholipids and the subsequent modification of LDL, we incubated 250 microM phosphatidylcholine with 10 microM ferrous sulfate and 50 microM ascorbic acid in 10 mM Tris (pH 7.0). After 18 h at 37 degrees C, significant amounts of thiobarbituric acid reactive substances (TBARS) were formed. The inclusion of LDL (100 micrograms protein/ml) elevated the TBARS and increased the electrophoretic mobility of the lipoprotein. LDL treated with iron and ascorbate in the absence of phosphatidylcholine did not result in the modification of this lipoprotein. LDL that was incubated with phosphatidylcholine, iron and ascorbate was found to be metabolized by macrophages to a far greater extent than native LDL or LDL treated with phosphatidylcholine alone. Probucol (10 microM) inhibited the LDL modification process. These results demonstrate that while iron and ascorbate cannot oxidize LDL directly, the addition of phosphatidylcholine to these initiators of lipid peroxidation can mediate and lead to the modification of LDL.

Mesothelin is overexpressed in the vast majority of ductal adenocarcinomas of the pancreas: identification of a new pancreatic cancer marker by serial analysis of gene expression (SAGE).

PURPOSE: Effective new markers of pancreatic carcinoma are urgently needed. In a previous analysis of gene expression in pancreatic adenocarcinoma using serial analysis of gene expression (SAGE), we found that the tag for the mesothelin mRNA transcript was present in seven of eight SAGE libraries derived from pancreatic carcinomas but not in the two SAGE libraries derived from normal pancreatic duct epithelial cells. In this study, we evaluate the potential utility of mesothelin as a tumor marker for pancreatic adenocarcinoma. EXPERIMENTAL DESIGN: Mesothelin mRNA expression was evaluated in pancreatic adenocarcinomas using reverse-transcription PCR (RT-PCR) and in situ hybridization, whereas mesothelin protein expression was evaluated by immunohistochemistry. RESULTS: Using an online SAGE database (http://www.ncbi.nlm.gov/SAGE), we found the tag for mesothelin to be consistently present in the mesothelioma, ovarian cancer, and pancreatic cancer libraries but not in normal pancreas libraries. Mesothelin mRNA expression was confirmed by in situ hybridization in 4 of 4 resected primary pancreatic adenocarcinomas and by RT-PCR in 18 of 20 pancreatic cancer cell lines, whereas mesothelin protein expression was confirmed by immunohistochemistry in all 60 resected primary pancreatic adenocarcinomas studied. The adjacent normal pancreas in these 60 cases did not label, or at most only rare benign pancreatic ducts showed weak labeling for mesothelin. CONCLUSIONS: Mesothelin is a new marker for pancreatic adenocarcinoma identified by gene expression analysis. Mesothelin overexpression in pancreatic adenocarcinoma has potential diagnostic, imaging, and therapeutic implications.

The Study of Optical Properties as Glass Composition of Bi2O3-Based Glass/Phosphor Mixed Paste.

Recently, White light emitting diodes (WLEDs) have been studied because of many advantages such as lower energy consumption, fast response, high brightness. Glass frit has been interested in LED packages due to their superior properties such as long-term stability and permeability. To maximize the LED light emission characteristic, the glass frit was required a low firing temperature and high refractive index. We selected the bismuth-based glass due to their low melting and high refractive index. This study was investigated characteristics of glass according to the influence of the glass within Bi2O3 content and this glass characteristic change was studied the effects on the optical properties of LED package structure. The properties changes of the glass frit affect the optical property of the mixed paste. With higher contents of Bi203 glass composition, the transmittance and emission intensity of the mixed paste was increased. These results suggest that the difference in refractive index between the phosphor and glass frit is minimized, the loss of light is minimized.

Expression and mutation analyses of MKK4, a candidate tumour suppressor gene encoded by chromosome 17p, in human gastric adenocarcinoma.

Homozygous deletion or somatic mutations of mitogen-activated protein kinase kinase 4 (MKK4), a candidate tumour suppressor gene located at 17p11, have been observed in many types of human tumours. To explore the likelihood that MKK4 acts as a suppressor in gastric tumorigenesis, we examined the expression and mutation status of MKK4 in 144 gastric tissues and cell line specimens. Expression of the MKK4 transcript was easily detectable in all normal and benign tumour tissues and none of 102 primary carcinomas and cell lines showed an abnormal reduction in MKK4 expression. Expression levels of MKK4 transcript showed no cancer-specific reduction in 43 matched sets and did not correlate with stage, grade and histopathological types of the tumours. Western blot analysis also revealed that MKK4 protein expression in carcinoma tissues and cell lines is comparable to non-cancerous tissues. A significant loss of heterozygosity (LOH) was detected at telomeric markers of the MKK4, locus. However, no allelic deletion of the MKK4 gene or at the centromeric loci was identified. Moreover, no evidences for somatic mutations leading to amino acid substitutions or frameshifts of MKK4 were identified in the carcinoma tissues and cell lines, whereas a substantial fraction of the same set showed allelic loss or mutations of the TP53 gene located at 17p13, suggesting that LOH at telomeric loci or the TP53 locus might not extend into the MKK4 gene in gastric cancers. In this study, we also report the identification of a highly conserved MKK4 processed pseudogene, which shares 95% homology with the coding region of the functional MKK4 transcript. Collectively, our data demonstrate that genomic deletion or somatic mutation of MKK4 is infrequent in gastric cancers, suggesting that MKK4 might not be a critical target of genetic inactivation in gastric tumorigenesis.

Cholesteryl esterase-treated LDL augments oxidized LDL-mediated cholesteryl ester deposition in mouse peritoneal macrophages.

Arterial unesterified cholesterol, phospholipid particles have been isolated from atherosclerotic lesions and characterized. However, the role of these 'liposomes' in macrophage foam cell formation is unclear. Recently, LDL, after trypsin and cholesteryl esterase treatment (T/CE LDL), was shown to have physical properties similar to the unesterified cholesterol, phospholipid particles isolated from atherosclerotic lesions. Yet, when mouse peritoneal macrophages were incubated with these model particles in culture medium (DMEM and 5% LPDS), only an insignificant accumulation of cellular cholesteryl esters was observed. Previously, we demonstrated that complex formation between unesterified cholesterol, phosphatidylcholine liposomes and cupric sulfate-oxidized LDL dramatically enhances the ability of the liposomes to augment cellular cholesterol accretion (Greenspan P, Yu H, Mao F, Gutman RL. J Lipid Res 1997;38:101-109). When T/CE LDL, another cholesterol-rich phospholipid particle, was substituted for unesterified cholesterol phosphatidylcholine liposomes in our complex, mouse peritoneal macrophages accumulated a significant amount of both cellular unesterifed cholesterol (61 microg/mg cell protein) and cholesteryl esters (76 microg/mg cell protein) after 48 h of incubation. These results demonstrate again that the interaction of two cholesterol-bearing particles (T/CE LDL and oxidized LDL), which individually can not promote significant cholesterol accumulation in cells, will, when combined, produce macrophage foam cells.

A neuron-specific gene transfer by a recombinant defective Sindbis virus.

We examined the possibility that Sindbis virus, an alpha virus with a single-stranded RNA genome, would be applied for neuronal gene transfer. The recombinant defective Sindbis viruses were constructed by replacing the structural genes of Sindbis virus with genes encoding beta-galactosidase (rdSind-lacZ) or enhanced green fluorescent protein (rdSind-EGFP). In neuron-glia cocultures prepared from the neocortex, hippocampus, and striatum, EGFP or beta-galactosidase was expressed selectively in neurons 24 h after infection with rdSind-EGFP or rdSind-lacZ. Most cortical neurons were infected with rdSind-lacZ at a multiplicity of infection (M.O.I.) of 5 while glial cells were little infected. In addition, transient neuron-specific expression of beta-galactosidase was observed near injection sites over the next 3 d following administration of rdSind-lacZ in adult rat. In the cortical neurons infected with rdSind-EGFP, treatment with NMDA induced neuritic blebs and cell body swelling in a Na+-dependent manner. Therefore, recombinant defective Sindbis viruses can be used as an efficient and selective vector for gene transfer into neurons and applied to investigate biological role of target genes delivered into neurons in vitro and in vivo.

Advances in agarose gel electrophoresis of serum lipoproteins.

Agarose gel electrophoresis has been extensively employed by researchers to gain a greater understanding of lipoprotein biology and its relationship to cardiovascular disease. Advances in this technique have been made in the visualization and quantitation of separated lipoproteins, in the use of agarose gel electrophoresis for detection and quantitation of apolipoproteins of the separated lipoproteins, and in the detection of lipoprotein heterogeneity. Agarose gel electrophoresis has been employed for two-dimensional electrophoretic analysis of lipoproteins as well as in several different methods which probe the immunological properties of lipoproteins. Agarose gel electrophoresis has thus become an important tool in the study of serum lipoproteins in both clinical and basic science laboratories.