Polyvinylidene Difluoride Filter Research Articles

Relationships among bacterial cell size, diversity, and taxonomy in rumen.

The rumen microbial community plays a crucial role in the digestion and metabolic processes of ruminants. Although sequencing-based studies have helped reveal the diversity and functions of bacteria in the rumen, their physiological and biochemical characteristics, as well as their dynamic regulation along the digestion process in the rumen, remain poorly understood. Addressing these gaps requires pure culture studies to demystify the intricate mechanisms at play. Bacteria exhibit morphological differentiation associated with different species. Based on the difference in size or shape of microorganisms, size fractionation by filters with various pore sizes can be used to separate them. In this study, we used polyvinylidene difluoride filters with pore sizes of 300, 120, 80, 40, 20, 8, 6, 2.1, and 0.6 μm. Bacterial suspensions were successively passed through these filters for the analysis of microbial population distribution using 16S rRNA gene sequences. We found that bacteria from the different pore sizes were clustered into four branches (> 120 μm, 40-120 μm, 6-20 μm, 20-40 μm, and < 0.6 μm), indicating that size fractionation had effects on enriching specific groups but could not effectively separate dominant groups by cell size alone. The species of unclassified Flavobacterium, unclassified Chryseobacterium, unclassified Delftia, Methylotenera mobilis, unclassified Caulobacteraceae, unclassified Oligella, unclassified Sphingomonas, unclassified Stenotrophomonas, unclassified Shuttleworthia, unclassified Sutterella, unclassified Alphaproteobacteria, and unclassified SR1 can be efficiently enriched or separated by size fractionation. In this study, we investigated the diversity of sorted bacteria populations in the rumen for preliminary investigations of the relationship between the size and classification of rumen bacteria that have the potential to improve our ability to isolate and culture bacteria from the rumen in the future.

(Invited) Three-Dimensional Filter of Polyvinylidene Difluoride (PVDF) Implanted Rice Husk-Derived Silicon Dioxide of Porous Solid-State Electrolytes

Lithium-ion batteries are the most promising energy storage devices in the battery technology field. They are widely used for numerous advantages, such as high energy density, fast charging, and being environmentally friendly. Lithium metal as an anode is highly attractive among different electrode materials because of its extraordinarily high specific capacity. However, lithium metal is prone to form dendrites during the battery charging process. Unfortunately, the conventional battery composed of flammable organic liquid electrolytes fails to suppress the lithium dendrite formation. The lithium dendrite thus penetrates the separator, causing battery short and bringing about explosions and catastrophes. Solid-state electrolytes (SSEs) have recently attracted much attention due to their higher energy density, wide electrochemical stability window, and longer cycle life. In addition, SSEs are mechanically more robust, suppressing the lithium dendrites formation. Therefore, replacing liquid electrolytes with solid-state electrolytes is an effective strategy to resolve safety concerns. To increase the mechanical strength of the SSEs, in this work, we report a three-dimensional filter-network-reinforced SSEs with a porous polyvinylidene difluoride (PVDF) filter host, polyethylene glycol (PEG) matrix with bistrifluoromethanesulfonimide lithium salt (LiTFSI) and rice husk-derived silicon dioxide (RHSiO2) as a passive filler. Rice husk-derived silicon dioxide possesses many superiorities, such as low-cost, easy processing, and non-toxicity. The mechanically robust PVDF filter helps to improve the mechanical property (tensile strength > 5 MPa) and prevents dendrite penetration, which raises thermal and mechanical stability. At room temperature, the ionic conductivity of the 5wt% SSE reach 1.110-4 S cm-1. The battery with 5wt% RHSiO2 SSE enabled stable cycling for over 300 hr during the Li plating and stripping process, demonstrating that the PVDF filter host SSEs with RHSiO2 is promising for the high-safety energy storage systems.

Sterilization performance comparison between an autosampler-ready microporous filter vial and a syringe-based filter

Herein we report that the Thomson Standard and eXtreme/FV® filter vials (0.2 μm polyvinylidene difluoride filter membrane) are as effective as gold standard microporous membrane-based syringe filters at removing bacteria, such as Klebsiella pneumonia, from media samples produced in the microbiological laboratory.

A filtration optimized on-line SPE–HPLC–MS/MS method for determination of three macrolide antibiotics dissolved and bound to suspended solids in surface water

In this work, a simple, automated multiresidue method consisting of on-line solid phase extraction coupled to liquid chromatography tandem mass spectrometry has been developed for the determination of three macrolide antibiotic residues – erythromycin, clarithromycin and azithromycin – included in the current Watch List (Decision 2018/840) of Water Framework Directive of the European Union in surface water. Due to the possible losses of target analytes, the effect of filtration on their concentrations was evaluated in terms of the filter material and pore size. Thus, glass fiber-nylon (0.45 μm and 0.22 μm pore sizes), 0.2 μm pore size nylon and 0.2 μm pore size polyvinylidene difluoride filters were used. As the Directive (2008/105/EC) prescribes the whole water monitoring, both the filtrate and solid fraction were analyzed. To estimate the analyte losses arisen from the adsorption on the solid phase, elution of the loaded filter with different aliquots of acetonitrile was also studied. The most suitable filter was the nylon of 0.2 μm pore size one, ensuring recoveries >80% for all three target analytes. By washing the filter with 2 mL acetonitrile, about 100% recovery rate was achieved for each macrolide in ultrapure water as well as river water spiked to 200 μg L−1 for each target analyte. The proposed sample pre-treatment protocol was successfully applied for the analysis of Danube River samples by using the internal standard calibration method. In one sample taken nearby a wastewater treatment plant, a relatively high concentration of clarithromycin (68 ± 0.4 ng L−1) was found.

Simultaneous identification of abused drugs, benzodiazepines, and new psychoactive substances in urine by liquid chromatography tandem mass spectrometry

A literature search reveals no studies concerning simultaneous identification of commonly abused drugs, benzodiazepines, and new psychoactive substances in urine by liquid chromatography tandem mass spectrometry (LC–MS/MS). We developed and validated an LC–MS/MS method for simultaneous identification of multiple abused drugs, benzodiazepines, and new psychoactive substances in urine from suspected drug abusers. The instrument was operated in multiple-reaction monitoring using an electrospray ionization mode. Chromatograms were separated using an ACE5 C18 column on a gradient of acetonitrile. After liquid–liquid extraction, samples were passed through a 0.22-μm polyvinylidene difluoride filter before injection into the LC–MS/MS. The limits of quantitation ranged from 0.5 ng/mL to 31.3 ng/mL. The linearity ranged from 0.5 ng/mL to 200 ng/mL. The precision results were below 15.4% (intraday) and 18.7% (interday). The intraday accuracy ranged from 85.9% to 121.0%; interday accuracy ranged from 66.1% to 128.7%. The proposed method was applied to 769 urine samples. The most common three drugs identified were ketamine, amphetamine, and opiates. The drug positive rate for one or more drugs was 79.6%. Our results demonstrate the suitability of the LC–MS/MS method for simultaneous identification of multiple abused drugs, benzodiazepines, and new psychoactive substances in urine.

Discoidin Domain Receptor 2–microRNA 196a–Mediated Negative Feedback against Excess Type I Collagen Expression Is Impaired in Scleroderma Dermal Fibroblasts

Systemic sclerosis (SSc) is characterized by excess collagen deposition in the skin, due to intrinsic transforming growth factor-β (TGF-β) activation. We tried to determine the expression and the role of discoidin domain receptor 2 (DDR2) in SSc. The expression of DDR2 mRNA and protein was significantly decreased in SSc dermal fibroblasts, which was recovered by knocking down TGF-β. The knockdown of DDR2 in normal fibroblasts induced microRNA-196a expression, which led to type I collagen downregulation, indicating that DDR2 itself has a negative effect on microRNA-196a expression and inducible effect on collagen expression. In SSc fibroblasts, however, the DDR2 knockdown did not affect TGF-β signaling and microRNA-196a expression. The microRNA-196a levels were significantly decreased in normal fibroblasts treated with TGF-β and in SSc fibroblasts. Taken together our data indicate that, in SSc fibroblasts, intrinsic TGF-β stimulation induces type I collagen expression, and also downregulates DDR2 expression. This probably acts as a negative feedback mechanism against excess collagen expression, as a decreased DDR2 expression is supposed to stimulate the microRNA-196a expression and further change the collagen expression. However, in SSc fibroblasts the microRNA-196a expression was downregulated by TGF-β signaling. DDR2-microRNA-196a pathway may be a previously unreported negative feedback system, and its impairment may be involved in the pathogenesis of SSc.

Characterization of the Substrate Specificity of Human Carboxypeptidase A4 and Implications for a Role in Extracellular Peptide Processing

CPA4 (carboxypeptidase A4) is a member of the metallocarboxypeptidase family. CPA4 was originally found in a screen of mRNAs up-regulated by sodium butyrate-induced differentiation of cancer cells. Further studies suggested a relation between CPA4 and prostate cancer aggressiveness. In the present study, we determined that CPA4 is secreted from cells as a soluble proenzyme (pro-CPA4) that can be activated by endoproteases, such as trypsin. Three complementary approaches were used to study the substrate specificity of CPA4; kinetic analysis was performed using a new series of chromogenic substrates and some biologically relevant peptides, the cleavage of synthetic peptides was tested individually, and the cleavage of a mixture of >100 mouse brain peptides was examined using a quantitative peptidomics mass spectrometry-based approach. CPA4 was able to cleave hydrophobic C-terminal residues with a preference for Phe, Leu, Ile, Met, Tyr, and Val. However, not all peptides with C-terminal hydrophobic residues were cleaved, indicating the importance of additional residues within the peptide. Aliphatic, aromatic, and basic residues in the P1 position have a positive influence on the cleavage specificity. In contrast, acidic residues, Pro, and Gly have a negative influence in the P1 position. Some of the peptides identified as CPA4 substrates (such as neurotensin, granins, and opioid peptides) have been previously shown to function in cell proliferation and differentiation, potentially explaining the link between CPA4 and cancer aggressiveness. Taken together, these studies suggest that CPA4 functions in neuropeptide processing and regulation in the extracellular environment.

3H]Epibatidine Photolabels Non-equivalent Amino Acids in the Agonist Binding Site of Torpedo and α4β2 Nicotinic Acetylcholine Receptors

Nicotinic acetylcholine receptor (nAChR) agonists, such as epibatidine and its molecular derivatives, are potential therapeutic agents for a variety of neurological disorders. In order to identify determinants for subtype-selective agonist binding, it is important to determine whether an agonist binds in a common orientation in different nAChR subtypes. To compare the mode of binding of epibatidine in a muscle and a neuronal nAChR, we photolabeled Torpedo alpha(2)betagammadelta and expressed human alpha4beta2 nAChRs with [(3)H]epibatidine and identified by Edman degradation the photolabeled amino acids. Irradiation at 254 nm resulted in photolabeling of alphaTyr(198) in agonist binding site Segment C of the principal (+) face in both alpha subunits and of gammaLeu(109) and gammaTyr(117) in Segment E of the complementary (-) face, with no labeling detected in the delta subunit. For affinity-purified alpha4beta2 nAChRs, [(3)H]epibatidine photolabeled alpha4Tyr(195) (equivalent to Torpedo alphaTyr(190)) in Segment C as well as beta2Val(111) and beta2Ser(113) in Segment E (equivalent to Torpedo gammaLeu(109) and gammaTyr(111), respectively). Consideration of the location of the photolabeled amino acids in homology models of the nAChRs based upon the acetylcholine-binding protein structure and the results of ligand docking simulations suggests that epibatidine binds in a single preferred orientation within the alpha-gamma transmitter binding site, whereas it binds in two distinct orientations in the alpha4beta2 nAChR.

Structural Basis for the Antiproliferative Activity of the Tob-hCaf1 Complex

The Tob/BTG family is a group of antiproliferative proteins containing two highly homologous regions, Box A and Box B. These proteins all associate with CCR4-associated factor 1 (Caf1), which belongs to the ribonuclease D (RNase D) family of deadenylases and is a component of the CCR4-Not deadenylase complex. Here we determined the crystal structure of the complex of the N-terminal region of Tob and human Caf1 (hCaf1). Tob exhibited a novel fold, whereas hCaf1 most closely resembled the catalytic domain of yeast Pop2 and human poly(A)-specific ribonuclease. Interestingly, the association of hCaf1 was mediated by both Box A and Box B of Tob. Cell growth assays using both wild-type and mutant proteins revealed that deadenylase activity of Caf1 is not critical but complex formation is crucial to cell growth inhibition. Caf1 tethers Tob to the CCR4-Not deadenylase complex, and thereby Tob gathers several factors at its C-terminal region, such as poly(A)-binding proteins, to exert antiproliferative activity.

ErbB-2 and Met Reciprocally Regulate Cellular Signaling via Plexin-B1

Sema4D-induced activation of plexin-B1 has been reported to evoke different and sometimes opposing cellular responses. The mechanisms underlying the versatility of plexin-B1-mediated effects are not clear. Plexin-B1 can associate with the receptor tyrosine kinases ErbB-2 and Met. Here we show that Sema4D-induced activation and inactivation of RhoA require ErbB-2 and Met, respectively. In breast carcinoma cells, Sema4D can have pro- and anti-migratory effects depending on the presence of ErbB-2 and Met, and the exchange of the two receptor tyrosine kinases is sufficient to convert the cellular response to Sema4D from pro- to anti-migratory and vice versa. This work identifies a novel mechanism by which plexin-mediated signaling can be regulated and explains how Sema4D can exert different biological activities through the differential association of its receptor with ErbB-2 and Met.

Trans geometric isomers of EPA decrease LXRα-induced cellular triacylglycerol via suppression of SREBP-1c and PGC-1β

Dietary mono- or di-trans fatty acids with chain lengths of 18-22 increase the risk of cardiovascular diseases because they increase LDL cholesterol and decrease HDL cholesterol in the plasma. However, the effects of trans isomers of PUFAs on lipid metabolism remain unknown. Dietary PUFAs, especially eicosapentaenoic acid (EPA) in marine oils, improve serum lipid profiles by suppressing liver X receptor alpha (LXRalpha) activity in the liver. In this study, we compared the effects of trans geometric isomers of eicosapentaenoic acid (TEPA) on triacylglycerol synthesis induced by a synthetic LXRalpha agonist (T0901317) with the effects of EPA in HepG2 cells. TEPA significantly decreased the amount of cellular triacylglycerol and the expression of mRNAs encoding fatty acid synthase, stearoyl-CoA desaturase-1, and glycerol-3-phosphate acyltransferase induced by T0901317 compared with EPA. However, there was no significant difference between the suppressive effect of TEPA or EPA on the expression of sterol-regulatory element binding protein-1c (SREBP-1c) induced by T0901317. We found that TEPA, but not EPA, decreased the mRNA expression of peroxisome proliferator-activated receptor gamma coactivator 1beta (PGC-1beta), which is a coactivator of both LXRalpha and SREBP-1. These results suggest that the hypolipidemic effect of TEPA can be attributed to a decrease not only in SREBP-1 but also in PGC-1beta expression.

The Novel WD-repeat Protein Morg1 Acts as a Molecular Scaffold for Hypoxia-inducible Factor Prolyl Hydroxylase 3 (PHD3)

Hypoxia-inducible factor-1 (HIF-1), a transcriptional complex composed of an oxygen-sensitive alpha- and a beta-subunit, plays a pivotal role in cellular adaptation to low oxygen availability. Under normoxia, the alpha-subunit of HIF-1 is hydroxylated by a family of prolyl hydroxylases (PHDs) and consequently targeted for proteasomal degradation. Three different PHDs have been identified, but the difference among their in vivo roles remain unclear. PHD3 is strikingly expressed by hypoxia, displays high substrate specificity, and has been identified in other signaling pathways. PHD3 may therefore hydroxylate divergent substrates and/or connect divergent cellular responses with HIF. We identified a novel WD-repeat protein, recently designated Morg1 (MAPK organizer 1), by screening a cDNA library with yeast two-hybrid assays. The interaction between PHD3 and Morg1 was confirmed in vitro and in vivo. We found seven WD-repeat domains by cloning the full-length cDNA of Morg1. By confocal microscopy both proteins co-localize within the cytoplasm and the nucleus and display a similar tissue expression pattern in Northern blots. Binding occurs at a conserved region predicted to the top surface of one propeller blade. Finally, HIF-mediated reporter gene activity is decreased by Morg1 and reduced to basal levels when Morg1 is co-expressed with PHD3. Suppression of Morg1 or PHD3 by stealth RNA leads to a marked increase of HIF-1 activity. These results indicate that Morg1 specifically interacts with PHD3 most likely by acting as a molecular scaffold. This interaction may provide a molecular framework between HIF regulation and other signaling pathways.

Tumor Cells Enhance Their Own CD44 Cleavage and Motility by Generating Hyaluronan Fragments

Hyaluronan (HA) is an extracellular matrix glycosaminoglycan that interacts with cell-surface receptors, including CD44. Although HA usually exists as a high molecular mass polymer, HA of a much lower molecular mass that shows a variety of biological activities can be detected under certain pathological conditions, particularly in tumors. We previously reported that low molecular weight HAs (LMW-HAs) of a certain size range induce the proteolytic cleavage of CD44 from the surface of tumor cells and promote tumor cell migration in a CD44-dependent manner. Here, we show that MIA PaCa-2, a human pancreatic carcinoma cell line, secreted hyaluronidases abundantly and generated readily detectable levels of LMW-HAs ranging from approximately 10- to 40-mers. This occurred in the absence of any exogenous stimulation. The tumor-derived HA oligosaccharides were able to enhance CD44 cleavage and tumor cell motility. Inhibition of the CD44-HA interaction resulted in the complete abrogation of these cellular events. These results are consistent with the concept that tumor cells generate HA oligosaccha-rides that bind to tumor cell CD44 through the expression of their own constitutive hyaluronidases. This enhances their own CD44 cleavage and cell motility, which would subsequently promote tumor progression. Such an autocrine/paracrine-like process may represent a novel activation mechanism that would facilitate and promote the malignant potential of tumor cells.

Cross-talk between the Two Divergent Insulin Signaling Pathways Is Revealed by the Protein Kinase B (Akt)-mediated Phosphorylation of Adapter Protein APS on Serine 588

The APS adapter protein is recruited to the autophosphorylated kinase domain of the insulin receptor and initiates the phosphatidylinositol 3-kinase (PI3K)-independent pathway of insulin-stimulated glucose transport by recruiting CAP and c-Cbl. In this study, we have identified APS as a novel substrate for protein kinase B/Akt using an antibody that exhibits insulin-dependent immunoreactivity with a phosphospecific antibody raised against the protein kinase B substrate consensus sequence RXRXX(pS/pT) and a phosphospecific antibody that recognizes serine 21/9 of glycogen synthase kinase-3alpha/beta. This phosphorylation of APS is observed in both 3T3-L1 adipocytes and transfected cells. The insulin-stimulated serine phosphorylation of APS was inhibited by a PI3-kinase inhibitor, LY290004, a specific protein kinase B (PKB) inhibitor, deguelin, and knockdown of Akt. Serine 588 of APS is contained in a protein kinase B consensus sequence for phosphorylation conserved in APS across multiple species but not found in other members of this family, including SH2-B and Lnk. Mutation of serine 588 to alanine abolished the insulin-stimulated serine phosphorylation of APS and prevented the localization of APS to membrane ruffles. A glutathione S-transferase fusion protein containing amino acids 534-621 of APS was phosphorylated by purified PKB in vitro, and mutation of serine 588 abolished the PKB-mediated phosphorylation of APS in vitro. Taken together, this study identifies APS as a novel physiological substrate for PKB and the first serine phosphorylation site on APS. These data therefore reveal the molecular cross-talk between the insulin-activated PI3-kinase-dependent and -independent pathways previously thought to be distinct and divergent.

A solid-phase immunoassay of protease-resistant prion protein with filtration blotting involving sodium dodecyl sulfate

The precise diagnosis for bovine spongiform encephalopathy (BSE) is crucial for preventing new transmission to humans. Several testing procedures are reported for determining protease-resistant prion protein in various tissues as a major hallmark of prion diseases such as BSE, scrapie, and Creutzfeldt–Jakob disease. However, contamination of materials from tissues or degradation of the specimens sometimes disturbs the accuracy of the assay. Here, we have developed a novel method for solid-phase immunoassay of the disease-specific conformational isoform, PrP Sc, using filtration blotting of protein in the presence of sodium dodecyl sulfate (SDS) followed by a filtration-based immunoassay with a single anti-prion protein antibody, together with the improved fractionation procedure involving high concentrations of surfactant/detergent. The SDS/heat treatment renders unfolded PrP Sc quantitative retention on a polyvinylidene difluoride filter and allows enhancement of the analyte signal with immunodetection; thus, all of the tested specimens are determined with 100% accuracy. In addition, the immunoassay is completed in approximately 1 h, indicating its usefulness not only for the screening of BSE specimens but probably also for the postmortem BSE diagnosis of fallen stock as the antibody recognizes the core part of PrP Sc. The solid-phase immunoassay method, including the filtration blotting with SDS, would be applicable to determining even more sensitively proteins other than PrP Sc, especially those having rigid conformations.

Glycosylation affects the protein stability and cell surface expression of Kv1.4 but Not Kv1.1 potassium channels. A pore region determinant dictates the effect of glycosylation on trafficking.

Kv1.1 and Kv1.4 potassium channels are plasma membrane glycoproteins involved in action potential repolarization. We have shown previously that glycosylation affects the gating function of Kv1.1 and that a pore region determinant of Kv1.1 and Kv1.4 affects their cell surface trafficking negatively or positively, respectively. Here we investigated the role of N-glycosylation of Kv1.1 and Kv1.4 on their protein stability, cellular localization pattern, and trafficking to the cell surface. We found that preventing N-glycosylation of Kv1.4 decreased its protein stability, induced its high partial intracellular retention, and decreased its cell surface protein levels, whereas it had little or no effect on these parameters for Kv1.1. Exchanging a trafficking pore region determinant between Kv1.1 and Kv1.4 reversed these effects of glycosylation on these chimeric channels. Thus it appeared that the Kv1.4 pore region determinant and the sugar tree attached to the S1-S2 linker showed some type of dependence in promoting proper trafficking of the protein to the cell surface, and this dependence can be transferred to chimeric Kv1.1 proteins that contain the Kv1.4 pore. Understanding the different trafficking programs of Kv1 channels, and whether they are altered by glycosylation, will highlight the different posttranslational mechanisms available to cells to modify their cell surface ion channel levels and possibly their signaling characteristics.

Planarisation of Patterned Aluminium/Diamond Surfaces for SAWdevices

AbstractA novel method for forming interdigitated electrodes for GHz Surface acoustic wave (SAW) devices on diamond using a damascene-like polishing technique is described. Low aspect ratio Al electrodes are recessed into the diamond substrates to create a near planar surface. This allows the deposition of higher quality oriented ZnO. CVD diamond samples were ion-beam etched with groove widths in the range 1-5μm to a depth of 80-120nm. Al (120nm thick) was sputter deposited on to the etched diamond and was polished using either (i) a solution of 0.05μm silica on a neoprene polishing cloth or (ii) using a ∼0.12mm thick PVDF (polyvinylidene difluoride) filter pad with colloidal solutions of silica. Using AFM scans and optical microscopy it was observed that the combination of method (i) with the small sample size resulted in significant hollowing-out of the Al electrodes and complete removal of Al from over 90% of the pad-interconnect area. The alternative technique in (ii) demonstrated far less Al removal and better uniformity in the electrode patterns.

Importance of Protein Kinase C Targeting for the Phosphorylation of Its Substrate, Myristoylated Alanine-rich C-kinase Substrate

We visualized the translocation of myristoylated alanine-rich protein kinase C substrate (MARCKS) in living Chinese hamster ovary-K1 cells using MARCKS tagged to green fluorescent protein (MARCKS-GFP). MARCKS-GFP was rapidly translocated from the plasma membrane to the cytoplasm after the treatment with phorbol ester, which translocates protein kinase C (PKC) to the plasma membrane. In contrast, PKC activation by hydrogen peroxide, which was not accompanied by PKC translocation, did not alter the intracellular localization of MARCKS-GFP. Non-myristoylated mutant of MARCKS-GFP was distributed throughout the cytoplasm, including the nucleoplasm, and was not translocated by phorbol ester or by hydrogen peroxide. Phosphorylation of wild-type MARCKS-GFP was observed in cells treated with phorbol ester but not with hydrogen peroxide, whereas non-myristoylated mutant of MARCKS-GFP was phosphorylated in cells treated with hydrogen peroxide but not with phorbol ester. Phosphorylation of both MARCKS-GFPs reduced the amount of F-actin. These findings revealed that PKC targeting to the plasma membrane is required for the phosphorylation of membrane-associated MARCKS and that a mutant MARCKS existing in the cytoplasm can be phosphorylated by PKC activated in the cytoplasm without translocation but not by PKC targeted to the membrane.

Analyte loss due to membrane filter adsorption as determined by high-performance liquid chromatography.

The phenomenon of membrane filter adsorption in high-performance liquid chromatography (HPLC) is investigated utilizing 16 brands of filters representing 3 polymeric materials: cellulose acetate (CA), nylon, and polyvinylidene difluoride in a variety of diameters (3, 4, 7, 13, and 25 mm). Sixteen compounds commonly encountered in drug preparations are selected as sample analytes and classified as acidic, basic, and neutral in chemical behavior. Six mobile phase/sample solvent mixtures are included: 3 with methanol-water and 3 with acetonitrile-water as major constituents. When using methanol as the mobile phase organic component, CA, nylon, and polyvinylidene difluoride (PVDF) filters exhibit negligible to moderate adsorption levels with regard to the neutral and basic drug compounds. The acidic drug test compounds are adsorbed by 50% of all 3 filter materials tested in methanol-water. In acetonitrile, neutral compounds are affected by 31.4%, basic compounds are affected by 47.0%, and acidic compounds are affected by 53.6% of the nylon and PVDF filters. CA is incompatible with acetonitrile and is excluded from the study with this solvent.

Protein Microarrays for Gene Expression and Antibody Screening

Proteins translate genomic sequence information into function, enabling biological processes. As a complementary approach to gene expression profiling on cDNA microarrays, we have developed a technique for high-throughput gene expression and antibody screening on chip-size protein microarrays. Using a picking/spotting robot equipped with a new transfer stamp, protein solutions were gridded onto polyvinylidene difluoride filters at high density. Specific purified protein was detected on the filters with high sensitivity (250 amol or 10 pg of a test protein). On a microarray made from bacterial lysates of 92 human cDNA clones expressed in a microtiter plate, putative protein expressors could be reliably identified. The rate of false-positive clones, expressing proteins in incorrect reading frames, was low. Product specificity of selected clones was confirmed on identical microarrays using monoclonal antibodies. Cross-reactivities of some antibodies with unrelated proteins imply the use of protein microarrays for antibody specificity screening against whole libraries of proteins. Because this application would not be restricted to antigen–antibody systems, protein microarrays should provide a general resource for high-throughput screens of gene expression and receptor–ligand interactions.