Analysis of glioma cell platinum response by metacomparison of two-dimensional chromatographic proteome profiles

Successful clinical development of cancer treatments is aided by the development of molecular markers that allow the identification of patients likely to respond. In the case of broadly cytotoxic drugs, such as the multinuclear series of platinum chemotherapeutic agents that we are evaluating for the treatment of glioma, one route to marker identification is proteomic profiling. We are using the two-dimensional chromatography system, the ProteomeLab PF2D, to compare proteomic profiles of glioma cells in culture before and after drug treatment. The existing software tools allowed the rapid identification of peaks increased by treatment of a given drug as compared with control untreated cells. To compare across these pairs, we developed new software, called the MetaComparison Tool (MCT). The MCT uses the chromatographic characteristics of peaks as identifiers, an approach that was validated by mass spectrometry of two independent isolations of a peak, from cells that were treated with two different platinum compounds. The MCT made it possible to rapidly query whether a given peak responded to more than one treatment and so allowed the identification of peaks that were specific to a given drug. As a result, this analysis greatly reduced the list of peaks whose isolation and downstream analysis by mass spectrometry is warranted, accelerating the search for protein markers of response.     

Novel Changes in NF-κB Activity during Progression and Regression Phases of Hyperplasia: ROLE OF MEK,ERK, AND p38*

Utilizing the Citrobacter rodentium-induced transmissible murine colonic hyperplasia (TMCH) model, we measured hyperplasia and NF-κB activation during progression (days 6 and 12 post-infection) and regression (days 20–34 post-infection) phases of TMCH. NF-κB activity increased at progression in conjunction with bacterial attachment and translocation to the colonic crypts and decreased 40% by day 20. NF-κB activity at days 27 and 34, however, remained 2–3-fold higher than uninfected control. Expression of the downstream target gene CXCL-1/KC in the crypts correlated with NF-κB activation kinetics. Phosphorylation of cellular IκBα kinase (IKK)α/β (Ser^176/180) was elevated during progression and regression of TMCH. Phosphorylation (Ser^32/36) and degradation of IκBα, however, contributed to NF-κB activation only from days 6 to 20 but not at later time points. Phosphorylation of MEK1/2 (Ser^217/221), ERK1/2 (Thr²⁰²/Tyr²⁰⁴), and p38 (Thr¹⁸⁰/Tyr¹⁸²) paralleled IKKα/β kinetics at days 6 and 12 without declining with regressing hyperplasia. siRNAs to MEK, ERK, and p38 significantly blocked NF-κB activity in vitro, whereas MEK1/2-inhibitor (PD98059) also blocked increases in MEK1/2, ERK1/2, and IKKα/β thereby inhibiting NF-κB activity in vivo. Cellular and nuclear levels of Ser⁵³⁶-phosphorylated (p65⁵³⁶) and Lys³¹⁰-acetylated p65 subunit accompanied functional NF-κB activation during TMCH. RSK-1 phosphorylation at Thr³⁵⁹/Ser³⁶³ in cellular/nuclear extracts and co-immunoprecipitation with cellular p65-NF-κB overlapped with p65⁵³⁶ kinetics. Dietary pectin (6%) blocked NF-κB activity by blocking increases in p65 abundance and nuclear translocation thereby down-regulating CXCL-1/KC expression in the crypts. Thus, NF-κB activation persisted despite the lack of bacterial attachment to colonic mucosa beyond peak hyperplasia. The MEK/ERK/p38 pathway therefore seems to modulate sustained activation of NF-κB in colonic crypts in response to C. rodentium infection.     

Chronic PKC-beta activation in HT-29 Cl.19a colonocytes prevents cAMP-mediated ion secretion by inhibiting apical membrane current generation

We investigated the effects of PKC-stimulating 12-deoxyphorbol 13-phenylacetate 20-acetate (DOPPA) and phorbol 12-myristate 13-acetate (PMA) phorbol esters on cAMP-dependent, forskolin (FSK)-stimulated, short-circuit Cl- current (ISC-cAMP) generation by colonocyte monolayers. These agonists elicited different actions depending on their dose and incubation time; PMA effects at the onset (<5 min) were independent of cAMP agonist and were characterized by transient anion-dependent transcellular and apical membrane ISC generation. DOPPA failed to elicit similar responses. Whereas chronic (24 h) exposure to both agents inhibited FSK-stimulated transcellular and apical membrane ISC-cAMP, the effects of DOPPA were more complex: this conventional PKC-beta-specific agonist also stimulated Ba2+-sensitive basolateral membrane-dependent facilitation of transcellular ISC-cAMP. PMA did not elicit a similar phenomenon. Prolonged exposure to high-dose PMA but not DOPPA led to apical membrane ISC-cAMP recovery. Changes in PKC alpha-, beta1-, gamma-, and epsilon-isoform membrane partitioning and expression correlated with these findings. PMA-induced transcellular ISC correlated with PKC-alpha membrane association, whereas low doses of both agents inhibited transcellular and apical membrane ISC-cAMP, increased PKC-beta1, decreased PKC-beta2 membrane association, and caused reciprocal changes in isoform mass. During the apical membrane ISC-cAMP recovery after prolonged high-dose PMA exposure, an almost-complete depletion of cellular PKC-beta1 and a significant reduction in PKC-epsilon mass occurred. Thus activated PKC-beta1 and/or PKC-epsilon prevented, whereas activated PKC-alpha facilitated, apical membrane ISC-cAMP. PKC-beta-dependent augmentation of transcellular ISC-cAMP at the level of the basolateral membrane demonstrated that transport events with geographically distinct subcellular membranes can be independently regulated by the PKC beta-isoform.     

Chronic PKC-beta2 activation in HT-29 Cl.19a colonocytes prevents cAMP-mediated ion secretion by inhibiting apical membrane CFTR targeting

We investigated the effects of chronically applied PKC-stimulating phorbol esters on subcellular CFTR expression and localization in polarized HT-29 Cl.19A monolayers. Modulation of PKC activity with the PKC-beta-specific agonist 12-deoxyphorbol 13-phenylacetate 20-acetate (DOPPA) or nonisoform-selective PMA altered monolayer CFTR immunofluorescence. A decrease in the CFTR signal within the luminal cellular pole was noted with both phorbol esters. Volumetric analysis of the intracellular CFTR signal revealed that both compounds promoted CFTR accumulation into punctate vesicle-like structures found adjacent to the cellular tight junction [labeled with zona occludens (ZO)-1 antibody], extending basally (DOPPA) into the cell. Puncta were more frequent with DOPPA and larger in size with PMA. DOPPA also promoted ZO-1 accumulation at tricellular corners associated with enhanced CFTR puncta number. The observed loss of CFTR immunofluorescence signal induced by low-dose PMA was related to CFTR sequestration into fewer cytoplasmic puncta and correlated with larger increases in PKC substrate phosphorylation. Both phorbol esters downregulated steady-state cellular CFTR mRNA levels by 70%. However, the effects of DOPPA and PMA were largely independent of CFTR biosynthesis: expression levels were 80-85% of control, and the glycosylation status of immunoprecipitated protein remained largely unchanged. Thus changes in cellular CFTR localization correlated with our companion study showing that PMA-induced inhibition of transcellular cAMP-dependent short-circuit current (ISC) was accompanied by cytoplasmic PKC-beta2 accumulation and modest activation of PKC-beta1 and PKC-epsilon. The inhibitory effect of DOPPA on ISC was related solely to increased cytoplasmic PKC-beta2 levels. Thus PKC-beta2 is hypothesized to participate in the regulation of CFTR apical plasma membrane targeting within the constitutive cellular biosynthetic pathway.     

Characterization of sialidase from bloodstream forms of Trypanosoma vivax

Sialidase (EC: 3.2.1.18) from Trypanosoma vivax (Agari Strain) was isolated from bloodstream forms of the parasite and purified to apparent electrophoretic homogeneity. The enzyme was purified 77-fold with a yield of 32% and co-eluted as a 66-kDa protein from a Sephadex G 110 column. The T. vivax sialidase was optimally active at 37 degrees C with an activation energy (E(a)) of 26.2 kJ mole(-1). The pH activity profile was broad with optimal activity at 6.5. The enzyme was activated by dithiothreitol and strongly inhibited by para-hydroxy mercuricbenzoate thus implicating a sulfhydryl group as a possible active site residue of the enzyme. Theenzyme hydrolysed Neu5Ac2,3lac and fetuin. It was inactive towards Neu5Ac2,6lac, colomic acid and the gangliosides GM1, and GDI. Initial velocity studies, for the determination of kinetic constants with fetuin as substrate gave a V(max) of 142.86 micromol h(-1) mg(-1) and a K(M) of 0.45 mM. The K(M) and V(max) with Neu5Ac-2,3lac were 0.17 mM and 840 micromole h(-1) mg(-1) respectively. The T. vivax sialidase was inhibited competitively by both 2,3 dideoxy neuraminic acid (Neu5Ac2,3en) and para-hydroxy oxamic acid. When ghost RBCs were used as substrates, the enzyme desialylated the RBCs from camel, goat, and zebu bull. The RBCs from dog, mouse and ndama bull were resistant to hydrolysis.     

Scaling and organization of electroencephalographic background activity and alpha rhythm in healthy young adults

The coexistence of the broad-band fluctuation and α rhythm of the brain dynamics is studied based on the zero-crossing property of the local electroencephalographic (EEG) recording in eyes closed and eyes open. A two-component zero-crossing scenario, consisting of a broad-band fractal and narrow-band rhythm components, is assumed. Scaling is found in the power law distribution p(τ) ~ τ ^−ν of the crossing time interval τ of the broad-band fluctuation. In α dominant brain state, the α rhythm interval L also exhibits scaling in the form of power law distribution: . Our main result is the relationship that characterizes the organization of these two prominent features of the brain dynamics. The possible role of self-organized criticality of punctuated equilibrium in this organization is discussed.     

Phosphoprotein enriched in astrocytes-15 kDa expression inhibits astrocyte migration by a protein kinase C delta-dependent mechanism

Phosphoprotein enriched in astrocytes-15 kDa (PEA-15), a phosphoprotein enriched in astrocytes, inhibits both apoptosis and proliferation in normal and cancerous cells. Here, analysis of PEA-15 expression in glioblastoma organotypic cultures revealed low levels of PEA-15 in tumor cells migrating away from the explants, regardless of the expression levels in the originating explants. Because glioblastomas are highly invasive primary brain tumors that can originate from astrocytes, we explored the involvement of PEA-15 in the control of astrocyte migration. PEA-15-/- astrocytes presented an enhanced motility in vitro compared with their wild-type counterparts. Accordingly, NIH-3T3 cells transfected by green fluorescent protein-PEA-15 displayed a reduced migration. Reexpression of PEA-15 restored PEA-15-/- astrocyte motility to wild-type levels. Pharmacological manipulations excluded a participation of extracellular signal-regulated kinase/mitogen-activated protein kinase, phosphatidylinositol 3-kinase/Akt, and calcium/calmodulin-dependent protein kinase II in this effect of PEA-15. In contrast, treatment by bisindolylmaleimide, G?6976, and rottlerin, and chronic application of phorbol 12-myristate 13-acetate and/or bryostatin-1 indicated that PKC delta mediated PEA-15 inhibition of astrocyte migration. PEA-15-/- astrocytes constitutively expressed a 40-kDa form of PKC delta that was down-regulated upon PEA-15 reexpression. Together, these data reveal a new function for PEA-15 in the inhibitory control of astrocyte motility through a PKC delta-dependent pathway involving the constitutive expression of a catalytic fragment of PKC delta.     

Precipitating and relieving factors of migraine versus tension type headache

ABSTRACT: BACKGROUND: To determine the differences of precipitating and relieving factors between migraine and tension type headache METHODS: This is a cross sectional study. We retrospectively reviewed the records of 250 migraine patients and 250 patients diagnosed as tension type headache from the specialized headache clinic in Dept. of Neurology, Dhaka Medical College Hospital. Data were collected through a predesigned questionnaire containing information on age, sex, social status and a predetermined list of precipitating and relieving factors. RESULTS: In this study, the female patients predominated (67%). Most of the patients were within 21--30 years age group (58.6%). About 58% of them belonged to middle class families. The common precipitating factors like stress, anxiety, activity, journey, reading, cold and warm were well distributed among both the migraine and tension type headache (TTH) patients. But significant difference was demonstrated for fatigue (p < 0.05), sleep deprivation (p < 0.05), sunlight (p < 0.01) and food (p < 0.05), which were common among migraineurs. In consideration of relieving factors of pain, different maneuvers were commonly tried by migraineurs and significant difference were observed for both analgesic drug and massage (p < 0.05), which relieved migraine headache. But maneuvers like sleep, rest and posture were used by both groups. CONCLUSION: The most frequent precipitating factors for headache appear to be identical for both migraine and TTH patients. Even though some factors like fatigue, sleep deprivation, sunlight and food significantly precipitate migraine and drug, massage are effective maneuver for relieving pain among migrianeurs.     

Human endometrial stem cell neurogenesis in response to NGF and bFGF

The potential of cell therapy is promising in nerve regeneration, but is limited by ethical considerations about the proper and technically safe source of stem cells. We report the successful differentiation of human EnSCs (endometrial stem cells) as a rich source of renewable and safe progenitors into high-efficiency cholinergic neurons. The extracellular signals of NGF (nerve growth factor) and bFGF (basic fibroblast growth factor) could induce cholinergic neuron differentiation. ChAT (choline acetyltransferase), MAP2 (microtubule associated protein 2) and NF-l (neurofilament L) increased after administration of bFGF and NGF to the EnSC cultures. trkC and FGFR2 (fibroblast growth factor receptor 2), which belong to the NGF and bFGF receptors respectively, were determined in populations of EnSCs. NGF, bFGF and their combination differentially influenced human EnSCs high efficiency differentiation. By inducing cholinergic neurons from EnSCs in a chemically defined medium, we could produce human neural cells without resorting to primary culture of neurons. This in vitro method provides an unlimited source of human neural cells and facilitates clinical applications of EnSCs for neurological diseases.     

Molecular Epidemiology of Influenza A(H1N1)pdm09 Viruses from Pakistan in 2009-2010

Background

In early 2009, a novel influenza A(H1N1) virus that emerged in Mexico and United States rapidly disseminated worldwide. The spread of this virus caused considerable morbidity with over 18000 recorded deaths. The new virus was found to be a reassortant containing gene segments from human, avian and swine influenza viruses.

Methods/Results

The first case of human infection with A(H1N1)pdm09 in Pakistan was detected on 18^th June 2009. Since then, 262 laboratory-confirmed cases have been detected during various outbreaks with 29 deaths (as of 31^st August 2010). The peak of the epidemic was observed in December with over 51% of total respiratory cases positive for influenza. Representative isolates from Pakistan viruses were sequenced and analyzed antigenically. Sequence analysis of genes coding for surface glycoproteins HA and NA showed high degree of high levels of sequence identity with corresponding genes of regional viruses circulating South East Asia. All tested viruses were sensitive to Oseltamivir in the Neuraminidase Inhibition assays.

Conclusions

Influenza A(H1N1)pdm09 viruses from Pakistan form a homogenous group of viruses. Their HA genes belong to clade 7 and show antigenic profile similar to the vaccine strain A/California/07/2009. These isolates do not show any amino acid changes indicative of high pathogenicity and virulence. It is imperative to continue monitoring of these viruses for identification of potential variants of high virulence or drug resistance.