Culture Lysates Research Articles

Cell density regulates in vitro activation of heart valve interstitial cells

Valve interstitial cells, the most prominent cell type in the heart valve, are activated and express α-smooth muscle actin in valve repair and in diseased valves. We hypothesize that cell density, time in culture, and the establishment of cell-cell contacts may be involved in regulating valve interstitial cell activation in vitro. To study cell density, valve interstitial cells were plated at passages 3 to 5, at a density of 17,000 cells/22 × 22 mm(2) coverslip, and grown for 1, 2, 4, 7, and 10 days. Valve interstitial cells were stained for α-smooth muscle actin and viewed under confocal microscopy to characterize the intensity of staining. To study time in culture, valve interstitial cells were plated at a 10-fold higher density to achieve similar growth densities over a shorter time period compared with valve interstitial cells plated at low density. α-Smooth muscle actin staining was compared at the same time points between those plated at high and low densities. To confirm valve interstitial cell activation as indicated by α-smooth muscle actin staining, valve interstitial cells were stained for cofilin at days 2, 5, 8, and 14 days postplating. To study the association of transforming growth factor β with valve interstitial cell activation with respect to cell density, valve interstitial cells were stained for α-smooth muscle actin and transforming growth factor β at 2, 4, 6, and 8 days postplating. To study the activation of the transforming growth factor β signaling pathway, valve interstitial cells were stained for pSmad2/3 at days 2, 4, 6, 8, 10, and 12 days postplating. To study cell contacts and activation, subconfluent and confluent cultures of valve interstitial cells were stained for β-catenin, N-cadherin, and α-smooth muscle actin. Also, whole-cell lysates of subconfluent and confluent valve interstitial cell cultures were probed by Western blot analysis for phospho-β-catenin at Ser33/37/Thr41, which is the form of β-catenin targeted for proteosomal degradation. The percentage valve interstitial cells with high-intensity α-smooth muscle actin staining decreases significantly between days 1 and 4, and at confluency, most cells show absent or low-intensity staining, regardless of time in culture. Similar results are obtained with cofilin staining. Transforming growth factor β and nuclear pSmad2/3 staining in valve interstitial cells decreases concurrently with valve interstitial cell activation as cell density increases. Examining β-catenin and N-cadherin staining, single valve interstitial cells show no cell-cell contact with strong cytoplasmic staining, with some showing nuclear staining of β-catenin, while confluent monolayers show strong staining of fully established cell-cell contacts, weak cytoplasmic staining, and absent nuclear staining. The presence of cell-cell contacts is associated with a decreased α-smooth muscle actin. The level of phospho-β-catenin at Ser33/37/Thr41 is lower in confluent cultures compared with low-density subconfluent valve interstitial cell cultures. Cell-cell contacts may inhibit valve interstitial cell activation, while absence of cell-cell contacts may contribute to activation.

Application of the major capsid protein as a marker of the phylogenetic diversity of Emiliania huxleyi viruses

Studies of the Phycodnaviridae have traditionally relied on the DNA polymerase (pol) gene as a biomarker. However, recent investigations have suggested that the major capsid protein (MCP) gene may be a reliable phylogenetic biomarker. We used MCP gene amplicons gathered across the North Atlantic to assess the diversity of Emiliania huxleyi-infecting Phycodnaviridae. Nucleotide sequences were examined across >6000 km of open ocean, with comparisons between concentrates of the virus-size fraction of seawater and of lysates generated by exposing host strains to these same virus concentrates. Analyses revealed that many sequences were only sampled once, while several were over-represented. Analyses also revealed nucleotide sequences distinct from previous coastal isolates. Examination of lysed cultures revealed a new richness in phylogeny, as MCP sequences previously unrepresented within the existing collection of E. huxleyi viruses (EhV) were associated with viruses lysing cultures. Sequences were compared with previously described EhV MCP sequences from the North Sea and a Norwegian Fjord, as well as from the Gulf of Maine. Principal component analysis indicates that location-specific distinctions exist despite the presence of sequences common across these environments. Overall, this investigation provides new sequence data and an assessment on the use of the MCP gene.

Measurement of plasma hydrogen sulfide in vivo and in vitro

The gasotransmitter hydrogen sulfide is known to regulate multiple cellular functions during normal and pathophysiological states. However, a paucity of concise information exists regarding quantitative amounts of hydrogen sulfide involved in physiological and pathological responses. This is primarily due to disagreement among various methods employed to measure free hydrogen sulfide. In this article, we describe a very sensitive method of measuring the presence of H2S in plasma down to nanomolar levels, using monobromobimane (MBB). The current standard assay using methylene blue provides erroneous results that do not actually measure H2S. The method presented herein involves derivatization of sulfide with excess MBB in 100mM Tris–HCl buffer (pH 9.5, 0.1mM DTPA) for 30min in 1% oxygen at room temperature. The fluorescent product sulfide-dibimane (SDB) is analyzed by RP-HPLC using an eclipse XDB-C18 (4.6×250mm) column with gradient elution by 0.1% (v/v) trifluoroacetic acid in acetonitrile. The limit of detection for sulfide-dibimane is 2 nM and the SDB product is very stable over time, allowing batch storage and analysis. In summary, our MBB method is suitable for sensitive quantitative measurement of free hydrogen sulfide in multiple biological samples such as plasma, tissue and cell culture lysates, or media.

Direct analysis and stability of methylated trivalent arsenic metabolites in cells and tissues

Chronic ingestion of water containing inorganic arsenic (iAs) has been linked to a variety of adverse health effects, including cancer, hypertension and diabetes. Current evidence suggests that the toxic methylated trivalent metabolites of iAs, methylarsonous acid (MAs(III)) and dimethylarsinous acid (DMAs(III)) play a key role in the etiology of these diseases. Both MAs(III) and DMAs(III) have been detected in urine of subjects exposed to iAs. However, the rapid oxidation of DMAs(III) and, to a lesser extent, MAs(III) in oxygen-rich environments leads to difficulties in the analysis of these metabolites in samples of urine collected in population studies. Results of our previous work indicate that MAs(III) and DMAs(III) are relatively stable in a reducing cellular environment and can be quantified in cells and tissues. In the present study, we used the oxidation state-specific hydride generation-cryotrapping-atomic absorption spectroscopy (HG-CT-AAS) to examine the presence and stability of these trivalent metabolites in the liver of mice and in UROtsa/F35 cells exposed to iAs. Tri- and pentavalent metabolites of iAs were analyzed directly (without chemical extraction or digestion). Liver homogenates prepared in cold deionized water and cell culture medium and lysates were stored at either 0 °C or -80 °C for up to 22 days. Both MAs(III) and DMAs(III) were stable in homogenates stored at -80 °C. In contrast, DMAs(III) in homogenates stored at 0 °C began to oxidize to its pentavalent counterpart after 1 day; MAs(III) remained stable for at least 3 weeks under these conditions. MAs(III) and DMAs(III) generated in UROtsa/F35 cultures were stable for 3 weeks when culture media and cell lysates were stored at -80 °C. These results suggest that samples of cells and tissues represent suitable material for the quantitative, oxidation state-specific analysis of As in laboratory and population studies examining the metabolism or toxic effects of this metalloid.

C-Jun N-Terminal Kinase (JNK) and p38 Play Different Roles in Age-Related Purkinje Cell Death in Murine Organotypic Culture

Several studies have shown that Purkinje cells die by apoptosis in organotypic slice cultures from postnatal 3-day-old (P3) mice. This cell death is age-dependent and has been proposed as indirect evidence for the programmed Purkinje cell death occurring in in vivo cerebellum. Here, we studied whether c-jun N-terminal kinase (JNK) and p38 kinase pathways contribute to the Purkinje cell death observed in cerebellar slice cultures obtained from P3 mice. Slice culture treatment with D-JNKI1 or SB203580, respectively inhibitors of JNK and p38 MAP kinases, results in a better survival of Purkinje cells. Interestingly, the combined treatment with the two inhibitors potentiated single treatment effects. These results suggest that p38 and JNK pathways might be differently implicated in this Purkinje cell death. Time course experiments found p38 activation immediately post-slicing, whereas JNK activation was detected only 2h after the culture. We hypothesize that p38 activation might be due to the "sliced condition," and JNK activation might be more specific to P3 age-dependent cell death. The study of JNK and p38 activation in cerebellar lysates from P0 slice culture confirmed JNK activation being specific for the P3 explants, whereas p38 is activated both from P0 and P3 cerebellar slice culture lysates. These results suggest that p38 is activated by the slicing, whereas JNK activation is related to developmental Purkinje cell death.

Identificación rápida de Histoplasma capsulatum en lisados de cultivo

Background Histoplasma capsulatum is the agent of histoplasmosis, a deep mycosis mainly afflicting immunocompromised patients. Rapid identification of the fungus isolated from clinical specimens allows timely administration of specific treatment. Aim To assess the ability of a dual PCR system targeting specific H. capsulatum DNA sites to identify fungal species in simple aqueous lysates from cultured fungi. Methods We analysed the performance of two independent PCR reactions designed to amplify fragments of 111 and 279 bp included in H. capsulatum-specific gene AgM. We used 248 H. capsulatum strains and 68 isolates of other fungal species. Reaction templates consisted of aqueous lysates of cultured fungi (either in mycelial or yeast phase) obtained after three cycles of boiling and immediate cooling at 0 °C. Selected strains were submitted to conventional DNA extraction and/or sequencing. Results Both PCR systems performed identically. Amplification from aqueous lysates was achieved from 239 H. capsulatum strains; the remaining 9 strains only showed specific bands when purified DNA was used as template. Of all other fungal species tested, only 2 Emmonsia crescens strains amplified H. capsulatum-specific bands and sequences of their amplified PCR products matched > 97% with H. capsulatum sequences. Total test time did not exceed 7 h with 96% sensitivity, 97% specificity and 99% positive predictive value. Conclusions The assay is fast, accurate and economical, and can be an alternative method for presumptive identification of H. capsulatum in simple culture lysates.

Analysis of two pharmacodynamic biomarkers using acoustic micro magnetic particles on the ViBE bioanalyzer

Pharmacodynamic responses to drug treatment are often used to confirm drug-on-target biological responses. Methods ranging from mass spectrometry to immunohistochemistry exist for such analyses. By far, the most extensively used methodologies employ antigen-specific antibodies for detection (at a minimum) and, in some cases, target quantitation as well. Using a novel frequency-modulating technology from BioScale called acoustic micro magnetic particle (AMMP) detection, two pathway biomarkers were chosen for pharmacodynamic analysis and compared with either AlphaScreen or LI-COR Western blot assays. For these studies, pharmacodynamic biomarkers for both proteasome and phosphoinositol 3-kinase inhibition were used. Our results show clearly that the BioScale technology is a robust and rapid method for measuring recombinant standards or endogenously derived proteins from both tissue culture and mouse xenograft tumor lysates. Moreover, the sensitivity obtained with the BioScale platform compares favorably with LI-COR Western blot and AlphaScreen technologies. Furthermore, the use of the ViBE Bioanalyzer eliminates the labor-intensive effort of Western blot analysis and is devoid of the optical and other endogenous interfering substances derived from lysates of xenograft tumors typically observed with AlphaScreen.

Efficacy of loop mediated isothermal amplification (LAMP) assay for the laboratory identification of Mycobacterium tuberculosis isolates in a resource limited setting

Current methods of TB diagnosis are time consuming and less suited for developing countries. The LAMP (loop mediated isothermal amplification) is a rapid method more suitable for diagnosis in resource limited settings and has been proposed as a viable test requiring further evaluation for use as a laboratory method as well. We evaluated two LAMP assays, using culture lysates of clinical sputum samples (from Southern India) and compared it to a proprietary multiplex PCR reverse-hybridization line probe assay (‘GenoType MTBC’ from HAIN Lifescience GmbH, Germany). The LAMP procedure was modified to suit the local conditions. The Mycobacterium tuberculosis specific LAMP assay (‘MTB LAMP’) showed sensitivity and specificity, of 44.7% and 94.4% respectively in a 60 min format, 85.7% and 93.9% respectively in a 90 min format and 91.7%, and 90.9% respectively in a 120 min format. The Mycobacteria universal LAMP assay (‘Muniv LAMP’) showed a sensitivity of 99.1%. The LAMP was shown to be a rapid and accessible assay for the laboratory identification of M. tuberculosis isolates. Initial denaturation of template was shown to be essential for amplification in unpurified/dilute samples and longer incubation was shown to increase the sensitivity. The need for modification of protocols to yield better efficacy in this scenario needs to be addressed in subsequent studies.

Chemoprevention Activity of Temu Mangga Extracts

The chemoprevention activity of temu mangga extracts was investigated by determination of antioxidant activity with a peroxidation number method and gluthatione-S-transferase (GST) activity in Chang medium culture and cell lysate (total GST activity). The results indicated that ethanol extract has a strong antioxidant activity. It is caused by the phenolic group in the ethanol extract. Treatment Chang cell culture with 7th and 4th ethanol fractions increased the GST activity when compared to the control. The total GST activity (cytosolic and microsomal) increased when Chang cell culture was treated with H2O2/Fe+2. The decrease of the total GST activity was observed when 7th and 4th ethanol fractions were supplemented with H2O2/Fe+2 compared to the cell culture receiving H2O2/Fe+2 only.

Regulation of the Matriptase-Prostasin Cell Surface Proteolytic Cascade by Hepatocyte Growth Factor Activator Inhibitor-1 during Epidermal Differentiation

Matriptase, a membrane-tethered serine protease, plays essential roles in epidermal differentiation and barrier function, largely mediated via its activation of prostasin, a glycosylphosphatidylinositol-anchored serine protease. Matriptase activity is tightly regulated by its inhibitor hepatocyte growth factor activator inhibitor-1 (HAI-1) such that free active matriptase is only briefly available to act on its substrates. In the current study we provide evidence for how matriptase activates prostasin under this tight control by HAI-1. When primary human keratinocytes are induced to differentiate in a skin organotypic culture model, both matriptase and prostasin are constitutively activated and then inhibited by HAI-1. These processes also occur in HaCaT human keratinocytes when matriptase activation is induced by exposure of the cells to a pH 6.0 buffer. Using this acid-inducible activation system we demonstrate that prostatin activation is suppressed by matriptase knockdown and by blocking matriptase activation with sodium chloride, suggesting that prostatin activation is dependent on matriptase in this system. Kinetics studies further reveal that the timing of autoactivation of matriptase, prostasin activation, and inhibition of both enzymes by HAI-1 binding are closely correlated. These data suggest that, during epidermal differentiation, the matriptase-prostasin proteolytic cascade is tightly regulated by two mechanisms: 1) prostasin activation temporally coupled to matriptase autoactivation and 2) HAI-1 rapidly inhibiting not only active matriptase but also active prostasin, resulting in an extremely brief window of opportunity for both active matriptase and active prostasin to act on their substrates.

Abstract A26: Elevations of intracellular kinases AKT1, GSK3β, JNK1, JNK2, and ERK2 in primary breast cancer tissue quantified with a novel multipanel of single-molecule immunoassays

Abstract Purpose: The MAPK and PI3K signaling cascades are critical pathways in malignant transformation, tumor progression and multidrug resistance (MDR) of human cancers. Elucidation of the relative contributions of distinct kinases is made difficult due to many factors, including prevalence of multiple isoforms, posttranslational modifications, synergistic activation and pathway crosstalk. Clinical investigations into the role of phosphorylation of specific kinases are further hindered by the lack of ultrasensitive assay technology with requisite isoform- and phophorylation-state specificity. Improved assays are necessary to determine clinical relevance and potential diagnostic utility with a high degree of specificity. Methods: Adjacent normal and primary breast cancer tissue lysates were analyzed using a novel panel of ten immunoassays for quantifying the total and phosphorylated forms of AKT1, GSK3β, JNK1, JNK2 and ERK2. The ultrasensitive assay panel was developed for use with the Erenna® Immunoassay System (Singulex), which uses single molecule detection technology. Analytical sensitivity, interassay precision, linearity, and specificity were determined and compared to corresponding ELISA assays. The novel assay panel was validated in HeLa and Jurkat cell lysates, region of linearity was determined, and phospho-specificity of each assay was confirmed. All assays were validated for use in human plasma, cell culture lysates, and human tissue specimens. Results: Testing in matched breast cancer tissue lysates showed that all total kinase concentrations (tAKT1, tGSK3β, tERK2, tJNK1 and tJNK2) were found to be elevated in primary tumors compared to surrounding adjacent normal tissue, however concentrations of phosphorylated forms (pAKT1, pGSK3β, pERK2, pJNK1 and pJNK2) were not. All ten novel assays displayed exceptional linearity (R2=0.99) and precision (%CV 4.1 – 9.6), and were significantly more sensitive than the corresponding ELISA assay method based upon sample endpoint dilutions. The detection limits of the tAKT1, pAKT1, tGSK3β, pGSK3β, pERK2, tJNK2 and pJNK2 assays were all ≤0.3 pg/mL, while the detection limit of tERK2 was < 2pg/mL, tJNK1 was 4.5 pg/mL, and pJNK1 was 7 pg/mL. The LLoQ of each assay in the panel was sufficient for quantification from a minimum of approximately 100 to 200 cells, with exception of the total JNK1 assay, which required a minimum of approximately 800 cells in order to be quantifiable. Conclusions: We show that significant elevations in total kinase concentrations of tAKT1, tGSK3β, tERK2, tJNK1 and tJNK2 can be quantified in primary tumors compared to surrounding adjacent tissue using a panel of isoform- and phosphorylation-specific single molecule immunoassays. We have further demonstrated that assays in this panel can quantify analytes from as little as 100 cells, and exhibit superior analytical performance over ELISA based methods. Thus, these novel assays greatly improve upon pre-existing ELISA technology that is limited by assay sensitivity, providing a new tool for clinical investigations of the MAPK and PI3K signaling cascades. Citation Information: Clin Cancer Res 2010;16(14 Suppl):A26.

Angiotensin II signalling regulates skeletal muscle growth and myoblast chemotaxis

The regulatory role of angiotensin II (Ang II) in mediating skeletal muscle hypertrophy is largely unknown. We examined the impact of Ang II on myoblast chemotaxis using transwell assays. Exogenous treatment of Ang II significantly increased the migratory capacity of both C2C12 and primary myoblasts by 30 and 43% respectively, while Ang II receptor knockout myoblasts (AT1a−/−) demonstrated a significant attenuation of basal migration (38% of control) and did not respond to exogenous Ang II treatment. Ang II appeared to increase migration by inducing MMP2 activity as gelatin zymography analysis of cell culture media and lysates revealed a ~40% increase in gelatinase activity and pharmacological inhibition of MMP2 abolished the Ang II mediated increase in myoblast migration. Ang II also appeared to signal in a paracrine fashion to induce migration as 4h of cyclic mechanical stretch of myoblasts enhanced migration of co‐cultured myoblasts. To test the involvement of Ang II signaling during skeletal muscle regeneration in vivo, control and At1a−/− mice were subjected to cardiotoxin injury. In comparison to controls, at 14 and 21 days following injury, At1a−/− mice demonstrated 34 and 25% reductions in cross sectional area respectively. Taken together, these results demonstrate that Ang II modulates skeletal muscle fibre growth by regulating myoblast chemotaxis through MMP2.

Primary Cell Cultures from Human Renal Cortex and Renal-Cell Carcinoma Evidence a Differential Expression of Two Spliced Isoforms of Annexin A3

Primary cell cultures from renal cell carcinoma (RCC) and normal renal cortex tissue of 60 patients have been established, with high efficiency (more than 70%) and reproducibility, and extensively characterized. These cultures composed of more than 90% of normal or tumor tubular cells have been instrumental for molecular characterization of Annexin A3 (AnxA3), never extensively studied before in RCC cells although AnxA3 has a prognostic relevance in some cancer and it has been suggested to be involved in the hypoxia-inducible factor-1 pathway. Western blot analysis of 20 matched cortex/RCC culture lysates showed two AnxA3 protein bands of 36 and 33 kDa, and two-dimensional Western blot evidenced several specific protein spots. In RCC cultures the 36-kDa isoform was significantly down-regulated and the 33-kDa isoform up-regulated. Furthermore, the inversion of the quantitative expression pattern of two AnxA3 isoforms in tumor cultures correlate with hypoxia-inducible factor-1alpha expression. The total AnxA3 protein is down-regulated in RCC cultures as confirmed also in tissues by tissue microarray. Two AnxA3 transcripts that differ for alternative splicing of exon III have been also detected. Real-time PCR quantification in 19 matched cortex/RCC cultures confirms the down-regulation of longer isoform in RCC cells. The characteristic expression pattern of AnxA3 in normal and tumor renal cells, documented in our primary cultures, may open new insight in RCC management.

Isolation and characterization of bacteriophage ΦBP fromPaenibacillus polymyxaCCM 7400

A bacteriophage PhiBP infecting Paenibacillus polymyxa CCM 7400 was isolated from culture lysate. Electron microscopy of lysate samples revealed the presence of bacteriophage particles with polyhedral heads 56 nm in diameter and flexible noncontractile tails 144 nm in length. The profile of PhiBP structural proteins resembles that of other bacteriophages. The PhiBP genome consists of double-stranded DNA of 43-kbp size. Homology search of sequenced DNA fragments from EcoRI digest revealed regions with significant similarity to other known bacteriophage genes. Regions similar to phage terminase genes were identified within the 1.2-kbp fragment. Three lytic genes, two holin genes and one endolysin gene were identified within the 2.5-kbp fragment. We tested the isolates of P. polymyxa CCM 7400 for the presence of phage DNA on bacterial chromosome using PCR amplification with primers derived from proposed terminase and holin gene sequences. We confirmed the presence of PhiBP DNA on P. polymyxa chromosome by Southern hybridization. The bacteriophage PhiBP was capable of causing lysis of a P. polymyxaPhiBP lysogen despite the presence of the phage DNA on bacterial chromosome. Therefore, we concluded that PhiBP was a virulent mutant phage.

Expression plasmids and production of EGFP in stably transfected Acanthamoeba

New plasmids containing the TATA-Binding Protein (TBP), TBP Promoter Binding Factor (TPBF) or Glyceraldehyde Phosphate Dehydrogenase (GAPDH) gene promoters from Acanthamoeba castellanii are described. The promoters for Acanthamoeba TPBF and GAPDH genes were used to drive constitutive expression of enhanced green fluorescent protein (EGFP) in stably transfected Acanthamoeba. Based initially on fluorescence microscopy and SDS–PAGE analysis of EGFP, both promoters produce robust expression of EGFP, with the highest level obtained from the GAPDH gene promoter in cells grown in low concentrations of neomycin G418. Purification of EGFP from lysates of 22-ml cultures by conventional chromatography yielded approximately 1.1 mg of EGFP, a value that extrapolates to 50 mg per liter of cell culture. The results suggest that Acanthamoeba is a useful cost-effective system for the production of recombinant proteins.

A three‐way proteomics strategy allows differential analysis of yeast mitochondrial membrane protein complexes under anaerobic and aerobic conditions

To investigate the effect of anaerobiosis on the Saccharomyces cerevisiae mitochondrial proteome and the formation of respiratory chain and other protein complexes, we analyzed mitochondrial protein extracts that were enriched from lysates of aerobic and anaerobic steady-state chemostat cultures. We chose an innovative approach in which native mitochondrial membrane protein complexes were separated by 1-D blue native PAGE, which was combined with quantitative analysis of each complex subunit using stable isotope labeling. LC-FT(ICR)-MS/MS analysis was applied to identify and quantify the mitochondrial proteins. In addition, to establish if changes in mitochondrial complex composition occurred under anaerobiosis, we investigated the 1-D blue native PAGE protein migration patterns by Pearson correlation analysis. Surprisingly, we discovered that under anaerobic conditions, where the yeast respiratory chain is not active, the respiratory chain supercomplexes, such as complex V dimer, complex (III)(2)(IV)(2) and complex (III)(2)(IV) were still present, although at reduced levels. Pearson correlation analysis showed that the composition of the mitochondrial complexes was unchanged under aerobic or anaerobic conditions, with the exception of complex II. In addition, this latter approach allowed screening for possible novel complex interaction partners, since for example protein Aim38p, with a yet unknown function, was identified as a possible component of respiratory chain complex IV.

Apolipoprotein E4 Domain Interaction Induces Endoplasmic Reticulum Stress and Impairs Astrocyte Function

Domain interaction, a structural property of apolipoprotein E4 (apoE4), is predicted to contribute to the association of apoE4 with Alzheimer disease. Arg-61 apoE mice, a gene-targeted mouse model specific for domain interaction, have lower brain apoE levels and synaptic, functional, and cognitive deficits. We hypothesized that domain interaction elicits an endoplasmic reticulum (ER) stress in astrocytes and an unfolded protein response that targets Arg-61 apoE for degradation. Primary Arg-61 apoE astrocytes had less intracellular apoE than wild-type astrocytes, and unfolded protein response markers OASIS (old astrocyte specifically induced substance), ATF4, and XBP-1 and downstream effectors were up-regulated. ER stress appears to cause global astrocyte dysfunction as glucose uptake was decreased in Arg-61 apoE astrocytes, and astrocyte-conditioned medium promoted neurite outgrowth less efficiently than wild-type medium in Neuro-2a cell cultures. We showed age-dependent up-regulation of brain OASIS levels and processing in Arg-61 apoE mice. ER stress and astrocyte dysfunction represent a new paradigm underlying the association of apoE4 with neurodegeneration.

Interaction of Rab31 and OCRL‐1 in oligodendrocytes: Its role in transport of mannose 6‐phosphate receptors

Rab31, a protein that we cloned from an oligodendrocyte cDNA library, is required for transport of mannose 6-phosphate receptors (MPRs) from the trans-Golgi network (TGN) to endosomes and for Golgi/TGN organization. Here we extend the knowledge of the mechanism of action of Rab31 by demonstrating its interaction with OCRL-1, a phosphatidylinositol 4,5-diphosphate 5-phosphatase (PI(4,5)P(2) 5-phosphatase) that regulates the levels of PI(4,5)P(2) and PI(4)P, molecules involved in transport and Golgi/TGN organization. We show that Rab31 interacts with OCRL-1 in a yeast two-hybrid system, GST-Rab31 pull-down experiments, and coimmunoprecipitation of OCRL-1 using oligodendrocyte culture lysates. Rab31 and OCRL-1 colocalize in the TGN, post-TGN carriers, and endosomes. Cation-dependent MPR (CD-MPR) is sorted to OCRL-1-containing carriers, but CD63 and vesicular stomatitis virus G (VSVG) are not. siRNA-mediated depletion of endogenous Rab31 causes collapse of the TGN apparatus and markedly decreases the levels of OCRL-1 in the TGN and endosomes. Our observations indicate that the role of Rab31 in the Golgi/TGN structure and transport of MPRs depends on its capability to recruit OCRL-1 to domains of the TGN where the formation of carriers occurs. The importance of our observations is highlighted by the fact that mutation of OCRL-1 causes demyelination in humans.

Affinity Capture Mass Spectrometry of Biomarker Proteins Using Peptide Ligands from Biopanning

Affinity capture mass spectrometry was used to isolate and ionize protein A from Staphylococcus aureus from both a commercial source and cell culture lysate using matrix assisted laser desorption/ionization (MALDI) mass spectrometry. Two surfaces are compared: gold surfaces with immunoglobulin G covalently immobilized and silica surfaces with a covalently bound small peptide discovered via biopanning. A detection limit of 2.22 bacterial cells/mL of culture fluid was determined for the immobilized peptide surfaces. This study emphasizes the ability to use peptide ligands to effectively capture a biomarker protein out of a complex mixture. This demonstrates the potential to use biopanning to generate capture ligands for a large variety of target proteins and subsequently detect the captured protein using MALDI mass spectrometry.

Quantification of volatile compounds in goat milk Jack cheese using static headspace gas chromatography

Goat milk Jack cheeses were manufactured with different levels of proteolytic endo- and exopeptidases from lysed bacterial cultures and aged for 30 wk. The aroma compounds that are potentially important in contributing the typical flavor of goat milk Jack cheese were quantified using static headspace gas chromatography. The concentrations of volatile compounds were evaluated every 6 wk throughout the aging period. Odor activity values of volatile compounds were calculated using the sensory threshold values reported in literature and their concentrations in Jack cheeses. Odor activity values of identified compounds were used to assess their potential contribution to the aroma of goat milk Jack cheeses. The odor activity values indicated that the ketones 2-hexanone, 2-heptanone, 2-nonanone, and 2,3-butanedione (diacetyl) were important odor-active compounds. The major odor-active acids found in this semi-hard goat milk cheese were butanoic, 2-methyl butanoic, pentanoic, hexanoic, and octanoic acids. Among the aldehydes, propanal and pentanal had high odor activity values and likely contributed to the aroma of this cheese. The concentrations of butanoic, pentanoic, hexanoic, heptanoic, octanoic, and nonanoic acids increased significantly in goat milk Jack cheese throughout aging. The extracted enzymes from lysed bacterial cultures that were added to the cheeses during manufacturing caused considerable increases in the concentrations of butanoic and hexanoic acids compared with the control. However, the lower concentration of peptidases resulted in an increased concentration of butanal, whereas more peptidases resulted in a lower concentration of 2-nonanone in goat milk Jack cheeses.