Effect of zeta potential on the performance of a ring-type electroosmotic mixer.

In order to achieve faster mixing, a new type of electrokinetic mixer with a T-type channel is introduced. The proposed mixer takes two fluids from different inlets and combines them into a single channel. The fluids then enter a mixing chamber with different inner and outer radii. Four microelectrodes are positioned on the outer wall of the mixing chamber. The electric potentials on the four microelectrodes are sinusoidal with respect to time and have various maximum voltages, zeta potentials and frequency values. The working fluid is water and each inlet has a different initial concentration values. The incompressible Navier-Stokes equation is solved in the channel, with a slip boundary condition on the inner and outer walls of the mixing chamber. The convection-diffusion equation is used to describe the concentration of the dissolved substances in the fluid. The pressure, concentration and flow fields in the channel are calculated and the results are graphically depicted for various flow and electric conditions.

Spinal epidural lubricant grease collection mimicking traumatic spinal epidural hematoma.

We report an unusual case of a patient with an epidural fluid collection with signal characteristics mimicking an epidural hematoma. The patient presented with myelopathy caused by thoracic spinal cord compression after a traumatic injury to the chest and back. The injury was caused by high-pressure injection of industrial-grade lubricant grease. This case demonstrates that cord compression can be caused by exogenous material in the setting of trauma that can mimic an acute epidural hematoma.

CEACAM5 and CEACAM6 are major target genes for Smad3-mediated TGF-beta signaling.

The carcinoembryonic antigen (CEAs) family consists of a large group of evolutionarily and structurally divergent glycoproteins. The transforming growth factor-beta (TGF-beta) signaling pathway has been implicated in the stimulation of CEA secretion in TGF-beta-sensitive colon cells, thereby possibly modulating cell adhesion and differentiation. However, the specific CEAs targeted by TGF-beta signaling or underlying mechanism of the expression of CEAs has not yet been clarified. In this study, we investigated the specific CEAs targeted by the TGF-beta signaling pathway. In nine human gastric cancer cell lines examined, TGF-beta-responsive cell lines showed positive expression of CEAs. Expression patterns of CEA proteins correlated well with the level of CEA (CEACAM5) and CEACAM6 transcripts in these cell lines, but CEACAM1 expression was not observed in all of these cells. To investigate the role of TGF-beta signaling in CEA expression, we selected two TGF-beta unresponsive gastric cancer cell lines; SNU638 cells that contain a mutation in the TGF-beta type II receptor and SNU484 cells that express low to undetectable level of the TGF-beta pathway intermediate protein, Smad3. Restoration of TGF-beta signaling in these cells induced expression of the CEAs and increased activity of both CEA (CEACAM5) and CEACAM6 promoters. CEA expression was observed in the epithelium of the stomach of wild-type mice, but was markedly decreased in Smad3 null mice. These findings suggest that CEA (CEACAM5) and CEACAM6 are major target genes for Smad3-mediated TGF-beta signaling.

TrkC binds to the type II TGF-beta receptor to suppress TGF-beta signaling.

Growing evidence suggests that overexpression of TrkC, a member of the Trk family of neurotrophin receptors, could drive tumorigenesis, invasion and metastatic capability in cancer cells. However, relatively little is known about the mechanism of TrkC-mediated oncogenesis. The TrkC gene is a partner of the Tel-TrkC (ETV6-NTRK3) chimeric tyrosine kinase, a potent oncoprotein expressed in tumors derived from multiple cell lineages. Recently, we have shown that ETV6-NTRK3 suppresses transforming growth factor-beta (TGF-beta) signaling by directly binding to the type II TGF-beta receptor (TbetaRII). Here, we report that expression of TrkC also suppresses TGF-beta-induced Smad2/3 phosphorylation and transcriptional activation. Silencing TrkC expression by small interfering RNA in the highly metastatic 4T1 mammary tumor cell line expressing endogenous TrkC significantly enhanced TGF-beta-induced Smad2/3 phosphorylation and restored TGF-beta growth inhibitory activity. In contrast, expression of TrkC in 67NR cells, in which TrkC is not expressed, suppressed TGF-beta transcriptional activation. Moreover, we show that TrkC directly binds to the TbetaRII, thereby preventing it from interacting with the type I TGF-beta receptor (TbetaRI). These results indicate that TrkC is an inhibitor of TGF-beta tumor suppressor activity.

Genomic organization, chromosomal localization and regulation of expression of the neuronal nuclear matrix protein NRP/B in human brain tumors.

The nuclear matrix and its role in cell physiology are largely unknown, and the discovery of any matrix constituent whose expression is tissue- and/or cell-specific offers a new avenue of exploration. Studies of the novel neuronal nuclear matrix protein, NRP/B, reveal that it is an early and highly specific marker of neuronal induction and development in vertebrates, since its expression is restricted mainly to the developing and mature nervous system. These studies also show that NRP/B is involved in neuronal differentiation. To further examine the structure-function of NRP/B, we have cloned and characterized the murine Nrp/b gene. The murine gene consists of four exons interrupted by three introns that span 7.6kb of DNA. The complete open reading frame is localized in exon 3, suggesting that NRP/B is highly conserved during evolution. Chromosomal analysis shows that NRP/B is localized to chromosome 13 in mouse and chromosome 5q12-13 in human. Since our previous studies demonstrated that NRP/B is expressed in primary hippocampal neurons but not in primary astrocytes, we have characterized NRP/B mRNA and protein expression in various brain cell lines and in human brain tumors. Abundant expression of NRP/B mRNA and protein was observed in human neuroblastoma cell lines (IMR32, SKN-MC, SKN-SH), in glioblastoma cell lines (A172, T98G, U87-MG, U118-MG, U138-MG, and U373-MG), in neuroglioma (H4) and astrocytoma cell lines (CCF-STTG1 and SW1088). Confocal analysis of NRP/B in U87-MG glioblastoma cells indicated nuclear localization of NRP/B. NRP/B expression was also observed in human primary brain tumors including glioblastoma multiformae and astrocytomas (total of five cases). These results suggest that NRP/B expression is upregulated in human brain tumors including glioblastomas and astrocytomas, while under normal conditions NRP/B expression is restricted to neurons. This study implicates a role for NRP/B in brain tumor development.

Glutamate-stimulated activation of DNA synthesis via mitogen-activated protein kinase in primary astrocytes: involvement of protein kinase C and related adhesion focal tyrosine kinase.

Glutamate is the major excitatory neurotransmitter in the CNS. Although its role in neurons has been studied extensively, little is known about its function in astrocytes. We studied the effects of glutamate on signaling pathways in primary astrocytes. We found that the tyrosine kinase related adhesion focal tyrosine kinase (RAFTK) is tyrosine phosphorylated in response to glutamate in a time- and dose-dependent manner. This phosphorylation was pertussis toxin (PTX) sensitive and could be attenuated by the depletion of Ca2+ from intracellular stores. RAFTK tyrosine phosphorylation was mediated primarily by class I/II metabotropic glutamate receptors and depends on protein kinase C (PKC) activation. Glutamate treatment of primary astrocytes also results in a significant increase in the activity of the mitogen-activated protein kinases [extracellular signal-related kinases 1/2 (ERK1/2)]. Like RAFTK phosphorylation, ERK1/2 activation is PTX sensitive and can be attenuated by the depletion of intracellular Ca2+ and by PKC inhibition, suggesting that RAFTK might mediate the glutamate-dependent activation of ERK1/2. Furthermore, we demonstrated that glutamate stimulation of primary astrocytes leads to a significant increase in DNA synthesis. Glutamate-stimulated DNA synthesis is PTX sensitive and can be inhibited by the MAP kinase kinase inhibitor PD98059, suggesting that in primary astrocytes, glutamate might signal via RAFTK and MAP kinase to promote DNA synthesis and cell proliferation.

Characterization of Mayven, a novel actin-binding protein predominantly expressed in brain.

The cytoskeleton plays an important role in neuronal morphogenesis. We have identified and characterized a novel actin-binding protein, termed Mayven, predominantly expressed in brain. Mayven contains a BTB (broad complex, tramtrack, bric-a-brac)/POZ (poxvirus, zinc finger) domain-like structure in the predicted N terminus and "kelch repeats" in the predicted C-terminal domain. Mayven shares 63% identity (77% similarity) with the Drosophila ring canal ("kelch") protein. Somatic cell-hybrid analysis indicated that the human Mayven gene is located on chromosome 4q21.2, whereas the murine homolog gene is located on chromosome 8. The BTB/POZ domain of Mayven can self-dimerize in vitro, which might be important for its interaction with other BTB/POZ-containing proteins. Confocal microscopic studies of endogenous Mayven protein revealed a highly dynamic localization pattern of the protein. In U373-MG astrocytoma/glioblastoma cells, Mayven colocalized with actin filaments in stress fibers and in patchy cortical actin-rich regions of the cell margins. In primary rat hippocampal neurons, Mayven is highly expressed in the cell body and in neurite processes. Binding assays and far Western blotting analysis demonstrated association of Mayven with actin. This association is mediated through the "kelch repeats" within the C terminus of Mayven. Depolarization of primary hippocampal neurons with KCl enhanced the association of Mayven with actin. This increased association resulted in dynamic changes in Mayven distribution from uniform to punctate localization along neuronal processes. These results suggest that Mayven functions as an actin-binding protein that may be translocated along axonal processes and might be involved in the dynamic organization of the actin cytoskeleton in brain cells.

Single-voxel proton MR spectroscopy of nonneoplastic brain lesions suggestive of a neoplasm.

BACKGROUND AND PURPOSE: MR spectroscopy is used to characterize biochemical components of normal and abnormal brain tissue. We sought to evaluate common histologic findings in a diverse group of nonneoplastic diseases in patients with in vivo MR spectroscopic profiles suggestive of a CNS neoplasm. METHODS: During a 2-year period, 241 patients with suspected neoplastic CNS lesions detected on MR images were studied with MR spectroscopy. Of these, five patients with a nonneoplastic diagnosis were identified retrospectively; a sixth patient without tissue diagnosis was added. MR spectroscopic findings consistent with a neoplasm included elevated choline and decreased N-acetylaspartate and creatine, with or without detectable mobile lipid and lactate peaks. RESULTS: The histologic specimens in all five patients for whom tissue diagnoses were available showed significant WBC infiltrates, with both interstitial and perivascular accumulations of lymphocytes, macrophages, histiocytes, and (in one case) plasma cells. Reactive astrogliosis was also prominent in most tissue samples. This cellular immune response was an integral component of the underlying disorder in these patients, including fulminant demyelination in two patients, human herpesvirus 6 encephalitis in one patient, organizing hematoma from a small arteriovenous malformation in one patient, and inflammatory pseudotumor in one patient. Although no histologic data were available in the sixth patient, neoplasm was considered unlikely on the basis of ongoing clinical and neuroradiologic improvement without specific therapy. CONCLUSION: Nonneoplastic disease processes in the CNS may elicit a reactive proliferation of cellular elements of the immune system and of glial tissue that is associated with MR spectroscopic profiles indistinguishable from CNS neoplasms with current in vivo MR spectroscopic techniques. Such false-positive findings substantiate the need for histologic examination of tissue as the standard of reference for the diagnosis of intracranial mass lesions.

Focal brain lesions: effect of single-voxel proton MR spectroscopic findings on treatment decisions.

PURPOSE: To determine the influence of single-voxel proton magnetic resonance (MR) spectroscopic findings on the treatment of patients suspected of having a brain tumor. MATERIALS AND METHODS: Medical records were reviewed in 78 patients who underwent MR spectroscopy for evaluation of a focal brain mass suspected of being neoplastic. MR spectroscopic findings were positive for neoplasm in 49 patients and negative in 29. Treatment with or without performance of biopsy was noted. In patients with positive findings who underwent irradiation or chemotherapy without biopsy and in patients with negative findings who were treated medically or followed up for interval changes, MR spectroscopy was classified as having a potential positive influence on treatment. In patients with positive findings with subsequently proved nonneoplastic lesions and in patients with negative findings with subsequently proved tumors, MR spectroscopy was classified as having a potential negative influence. RESULTS: MR spectroscopy in eight (16%) patients with positive findings and in 15 (52%) patients with negative findings had a potential positive influence on treatment. In two (3%) patients, MR spectroscopy had a potential negative influence. CONCLUSION: MR spectroscopy may play a beneficial role in the management of suspected brain tumors. Prospective studies are needed to test the effect of MR spectroscopy on clinical practice and to measure costs and benefits.

Characterization and chromosomal localization of PTP-NP-2, a new isoform of protein tyrosine phosphatase-like receptor, expressed on synaptic boutons.

Recently, there have been several reports describing the cloning and characterization of the novel family of protein tyrosine phosphatase-like receptor molecules (known as IA-2 and PTP-NP/PTP-IAR/IA-2beta/phogrin), which may act as autoantigens in diabetes. Here, we report the molecular characterization and chromosomal localization of a new isoform of this family in brain termed PTP-NP-2 (for PTP-NP tyrosine phosphatase isoform), and its function in rat primary hippocampal neurons. PTP-NP-2 has 48% identity to IA-2. The principal difference between PTP-NP-2 and PTP-NP is a 17-amino-acid insert near the N-terminus of PTP-NP that is absent in PTP-NP-2. Genomic DNA analysis indicates that the 17-amino-acid insert is coded by a separate exon, suggesting that both IA-2beta and PTP-NP-2 are isoforms arising by alternate splicing of the same gene. Reverse transcriptase-PCR revealed that both isoforms are present in human SH-SY5Y neuroblastoma cells. PTP-NP-2 mRNA expression is highly restricted, with a 5.5-kb specific transcript in human fetal and adult brain and 5.5 and 3. 8 kb in human adult pancreas. SH-SY5Y neuroblastoma and U87-MG glioblastoma cells showed specific transcripts of 5.5 and 3.8kb, respectively, indicating the existence of several isoforms of this molecule in the nervous system. The human gene encoding PTP-NP-2 was assigned to human chromosome 7q22-qter using Southern blot analysis of genomic DNAs from rodent/human somatic hybrid cell lines. Confocal microscopy analyses of rat primary hippocampal neurons revealed that PTP-NP-2 is abundantly expressed on synaptic boutons in primary neurons. Wild-type PTP-NP-2 showed no measurable tyrosine phosphatase activity using an in-vitro pNPP assay. Examination of the PTP-NP-2 catalytic consensus sequence revealed that this sequence differed from the typical tyrosine phosphatase-domain consensus sequence by an alanine to aspartate change (amino acid 930). Mutation of aspartate 930 to alanine produced a catalytically active enzyme, suggesting that native PTP-NP and its isoform PTP-NP-2 are catalytically inactive receptor protein tyrosine phosphatase homologues. Taken together, these results indicate that the tyrosine phosphatase PTP-NP-2 is a new isoform of PTP-NP tyrosine phosphatase, is expressed on synaptic boutons and may participate in the regulation of synaptic bouton endocytosis.

NRP/B, a novel nuclear matrix protein, associates with p110(RB) and is involved in neuronal differentiation.

The nuclear matrix is defined as the insoluble framework of the nucleus and has been implicated in the regulation of gene expression, the cell cycle, and nuclear structural integrity via linkage to intermediate filaments of the cytoskeleton. We have discovered a novel nuclear matrix protein, NRP/B (nuclear restricted protein/brain), which contains two major structural elements: a BTB domain-like structure in the predicted NH2 terminus, and a "kelch motif" in the predicted COOH-terminal domain. NRP/B mRNA (5.5 kb) is predominantly expressed in human fetal and adult brain with minor expression in kidney and pancreas. During mouse embryogenesis, NRP/B mRNA expression is upregulated in the nervous system. The NRP/B protein is expressed in rat primary hippocampal neurons, but not in primary astrocytes. NRP/B expression was upregulated during the differentiation of murine Neuro 2A and human SH-SY5Y neuroblastoma cells. Overexpression of NRP/B in these cells augmented neuronal process formation. Treatment with antisense NRP/B oligodeoxynucleotides inhibited the neurite development of rat primary hippocampal neurons as well as the neuronal process formation during neuronal differentiation of PC-12 cells. Since the hypophosphorylated form of retinoblastoma protein (p110(RB)) is found to be associated with the nuclear matrix and overexpression of p110(RB) induces neuronal differentiation, we investigated whether NRP/B is associated with p110(RB). Both in vivo and in vitro experiments demonstrate that NRP/B can be phosphorylated and can bind to the functionally active hypophosphorylated form of the p110(RB) during neuronal differentiation of SH-SY5Y neuroblastoma cells induced by retinoic acid. Our studies indicate that NRP/B is a novel nuclear matrix protein, specifically expressed in primary neurons, that interacts with p110(RB) and participates in the regulation of neuronal process formation.

Recent advances in MR spectroscopy expand its applications in neurologic disease.

MRS extends the diagnostic power of MRI by displaying the biochemical composition of a selected tissue or region. When MR imaging shows a lesion, the evaluation of the chemical composition by MRS can help determine whether biopsy, observation or medical treatment is indicated. It can save some patients from biopsy prior to radiation or chemotherapy. In the future, both the image information and the spatial distribution of chemical constituents throughout the brain will be displayed with techniques such as chemical shift imaging (CSI). MRS improves the accuracy of MRI diagnosis and prognosis. MRS is performed at many sites in the country and is reimbursed by many insurers. MRS has been approved by the AMA for a CPT-4 code for reimbursement.

Accuracy of single-voxel proton MR spectroscopy in distinguishing neoplastic from nonneoplastic brain lesions.

PURPOSE: To measure the accuracy of single-voxel, image-guided proton MR spectroscopy in distinguishing normal from abnormal brain tissue and neoplastic from nonneoplastic brain disease. METHODS: MR spectroscopy was performed at 0.5 T with the point-resolved spectroscopic pulse sequence and conventional postprocessing techniques. Subjects consisted of a consecutive series of patients with suspected brain neoplasms or recurrent neoplasia and 10 healthy adult volunteers. Fifty-five lesions in 53 patients with subsequently verified final diagnoses were included. Spectra were interpreted qualitatively by visual inspection by nonblinded readers (prospectively) with the benefit of prior clinical data and imaging studies, and by blinded readers (retrospectively). The nonblinded readers interpreted the spectra as diagnostic or not, and, if diagnostic, as neoplastic or nonneoplastic. The blinded readers classified the spectra as diagnostic or not, and, if diagnostic, as normal or abnormal and as neoplastic or nonneoplastic (when abnormal). The sensitivity, specificity, positive and negative predictive values, and accuracy were calculated from blinded and nonblinded MR spectroscopy interpretations. A receiver operator characteristic (ROC) curve analysis was performed on blinded MR spectroscopy interpretations. RESULTS: The diagnostic accuracy averaged across four blinded readers in differentiating patients from control subjects was .96, while the area under the aggregate (pooled interpretations) ROC curve approached unity. Accuracy in the nonblinded and blinded discrimination of neoplastic from nonneoplastic disease was .96 and .83, respectively. The area under the aggregate ROC curve in the blinded discrimination of neoplasm from nonneoplasm was .89. CONCLUSIONS: Image-guided proton spectra obtained at 0.5 T from patients with suspected neoplasia can be distinguished from spectra in healthy control subjects, and neoplastic spectra can be distinguished from nonneoplastic spectra with a high degree of diagnostic accuracy.

Solitary fibrous tumor of the paranasal sinuses: CT and MR appearance.

We describe the CT and MR appearance of a solitary fibrous tumor of the paranasal sinuses with intracranial invasion. The tumor was hypointense on T2-weighted MR images and had a large calcific component that proved to be reactive remodelling of native bone.

Transforming growth factor-beta regulation of retinoblastoma gene product and E2F transcription factor during cell cycle progression in mouse fibroblasts.

The mechanism by which transforming growth factor beta (TGF beta) exerts growth stimulatory effects was examined in C3H/10T1/2 mouse fibroblasts by study of cell cycle regulation of the retinoblastoma gene product (p110Rb) and transcriptional regulation of the p110Rb-associated transcription factor, E2F. Northern blotting analysis shows that TGF beta and/or epidermal growth factor (EGF) stimulate by three to sixfold the level of Rb mRNA which is also reflected by the increased levels of p110Rb. p110Rb becomes phosphorylated in mid-G1 and further phosphorylated at the G1/S transition. Hyperphosphorylation of p110Rb by TGF beta can be observed when cells are in S phase. TGF beta stimulates by three to fourfold the activity of cdk2 kinase consistent with the observed phosphorylation of p110Rb and also with the possibility that the kinase is involved in phosphorylating p110Rb close to the G1/S transition. Thus, TGF beta as a growth stimulator induces, as does EGF, the phosphorylation of p110Rb during cell cycle progression. Transient transfection of E2F promoter constructs was used to analyze the effect of TGF beta on the modulation of E2F-mediated transcription. The data revealed that TGF beta can stimulate wild-type adenoviral E2 promoter activity by 12-fold. Taken together, TGF beta-induced phosphorylation of p110Rb in mouse fibroblasts appears to exert a positive regulatory function upon genes that have a pivotal role in the G1/S transition of the cell cycle.

Transforming growth factor beta 1-induced delay of cell cycle progression and its association with growth-related gene expression in mouse fibroblasts.

TGF beta-induced cell cycle progression is relatively slower than that induced by EGF or PDGF-BB. Further, TGF beta delays EGF or PDGF-induced 5-phase entry in C3H 10T1/2 mouse fibroblasts. In accordance with this delay, the induction of mRNA level of 'immediate early genes' such as c-myc, c-fos, c-jun and junB by TGF beta has slower kinetics compared with those of EGF. TGF beta induces c-sis gene, suggesting possible involvement of secondary growth stimulation by PDGF-like proteins. However, anti-PDGF-AB antibody, which was inhibitory to FDGF-BB-induced [3H]thymidine incorporation, did not block TGF beta-induced DNA synthesis. These results first demonstrate that the delay of cell cycle progression by TGF beta is closely associated with the altered regulation of growth-related gene expression in fibroblasts.

Okadaic acid regulation of the retinoblastoma gene product is correlated with the inhibition of growth factor-induced cell proliferation in mouse fibroblasts.

Okadaic acid, a specific inhibitor of protein phosphatases 1 and 2A, was used to study the mechanism of action of transforming growth factor beta (TGF-beta) on cell cycle progression in C3H/10T1/2 mouse embryonic fibroblasts, where TGF-beta exerts a growth-stimulatory effect. Concentrations of okadaic acid as low as 5 nM inhibited TGF-beta (5 ng/ml)- or 10% serum-induced [3H]thymidine incorporation into postconfluent, quiescent cells. Further, these inhibitory effects were observed when okadaic acid was added as late as 10 hr after TGF-beta or serum stimulation. Since C3H/10T1/2 fibroblasts undergo the G1/S transition at 10-14 hr after TGF-beta and 8-12 hr after serum stimulation, these observations indicate that a phosphatase activity may be required for S-phase entry. In a parallel experiment, okadaic acid partially inhibited TGF-beta-induced [14C]leucine incorporation by 20-65%, depending upon the okadaic acid concentration. In conjunction with the effect of okadaic acid on DNA and protein synthesis, Western blot analysis indicated that okadaic acid inhibited phosphorylation of the retinoblastoma gene product and decreased its protein level, even when added 10 hr after TGF-beta or 8 hr after serum stimulation. These findings strongly suggest that protein phosphatases play a pivotal role for S-phase entry in mouse fibroblasts. Moreover, protein phosphatases may be required in the intermediate steps of TGF-beta or serum growth factor signal-transduction pathways for the stimulation of phosphorylation of the retinoblastoma protein, especially in late G1.