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Bladder cancer is the fourth most common cancer in men, and it is becoming a prevalent malignancy. Most of the regular clinical examinations are prompt evaluations with cystoscopy, renal function testing, which require high-precision instrument, well-trained operators, and high cost. In this study, a microfluidic paper-based analytical device (μPAD) was fabricated to detect nuclear matrix protein 22 (NMP22) and bladder cancer antigen (BTA) from the urine samples. Urine samples were collected from 11 bladder cancer patients and 10 well-beings as experiment and control groups, respectively, to verify the working efficiency of μPAD. A remarkable checkout efficiency of up to 90.91% was found from the results. Meanwhile, this method is feasible for home-based self-detection from urine samples within 10 min for the total process, which provides a new way for quick, economical, and convenient tumor diagnosis, prognosis evaluation, and drug response.

A complete mathematical model for electromigration in paper-based analytical devices is derived, based on differential equations describing the motion of fluids by pressure sources and EOF, the transport of charged chemical species, and the electric potential distribution. The porous medium created by the cellulose fibers is considered like a network of tortuous capillaries and represented by macroscopic parameters following an effective medium approach. The equations are obtained starting from their open-channel counterparts, applying scaling laws and, where necessary, including additional terms. With this approach, effective parameters are derived, describing diffusion, mobility, and conductivity for porous media. While the foundations of these phenomena can be found in previous reports, here, all the contributions are analyzed systematically and provided in a comprehensive way. Moreover, a novel electrophoretically driven dispersive transport mechanism in porous materials is proposed. Results of the numerical implementation of the mathematical model are compared with experimental data, showing good agreement and supporting the validity of the proposed model. Finally, the model succeeds in simulating a challenging case of free-flow electrophoresis in paper, involving capillary flow and electrophoretic transport developed in a 2D geometry. Fil: Schaumburg, Federico. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; Argentina Fil: Kler, Pablo Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Centro de Investigaciones en Métodos Computacionales. Universidad Nacional del Litoral. Centro de Investigaciones en Métodos Computacionales; Argentina Fil: Berli, Claudio Luis Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; Argentina

Microfluidic paper-based analytical devices (μPADs) allow user-friendly and portable chemical determinations, although they provide limited applicability due to insufficient sensitivity. Several approaches have been proposed to address poor sensitivity in μPADs, but they frequently require bulky equipment for power and/or read-outs. Universal serial buses (USB) are an attractive alternative to less portable power sources and are currently available in many common electronic devices. Here, USB-powered μPADs (USB μPADs) are proposed as a fusion of both technologies to improve performance without adding instrumental complexity. Two ITP USB μPADs were developed, both powered by a 5 V potential provided through standard USB ports. The first device was fabricated using the origami approach. Its operation was analyzed experimentally and numerically, yielding a two-order-of-magnitude sample focusing in 15 min. The second ITP USB μPAD is a novel design, which was numerically prototyped with the aim of handling larger sample volumes. The reservoirs were moved away from the ITP channel and capillary action was used to drive the sample and electrolytes to the separation zone, predicting 25-fold sample focusing in 10 min. USB μPADs are expected to be adopted by minimally-trained personnel in sensitive areas like resource-limited settings, the point-of-care and in emergencies. Fil: Schaumburg, Federico. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; Argentina Fil: Kler, Pablo Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Centro de Investigaciones en Métodos Computacionales. Universidad Nacional del Litoral. Centro de Investigaciones en Métodos Computacionales; Argentina Fil: Carrell, Cody S.. State University of Colorado - Fort Collins; Estados Unidos Fil: Berli, Claudio Luis Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; Argentina Fil: Henry, Charles S.. State University of Colorado - Fort Collins; Estados Unidos

Protein biomarkers often exist as degradation fragments in biological samples, and affinity agents derived using a purified protein may not recognize them, limiting their value for clinical diagnosis. Herein, we present a method to overcome this issue, by selecting aptamers against a degraded form of the toxin B protein, which is a marker for diagnosing toxigenic Clostridium difficile infections. This approach has led to isolation of a DNA aptamer that recognizes degraded toxin B, fresh toxin B, and toxin B spiked into human stool samples. DNA aptamers selected using intact recombinant toxin B failed to recognize degraded toxin B, which is the form present in stored stool samples. Using this new aptamer, we produced a simple paper-based analytical device for colorimetric detection of toxin B in stool samples, or in the NAP1 strain of Clostridium difficile. The combined aptamer-selection and paper-sensing strategy can expand the practical utility of DNA aptamers in clinical diagnosis.

Detection of biomarkers is essential for diagnosis and prognosis of diseases for healthcare teams to provide timely appropriate treatments. Reliable, inexpensive, and sensitive devices are desirable to serve this purpose. Paper-based analytical devices (PADs) have gained enormous attention during the past decade as point-of-care devices for biomarker detection due to their simplicity, portability, and biocompatibility. Importantly, the devices highly comply with the WHO "ASSURED" concept (i.e. Affordable, Sensitive, Specific, User-friendly, Rapid and Robust, Equipment free, and Deliverable to end-users). Thus, PADs could be sustainably alternative tools for biomarker detection, especially in resources-constraint areas where budget, skillful operators, and health infrastructure are limited. First, this review introduces overviews of biomarkers, their detection and brief history of PADs. Second, description of common fabrication and detection techniques in PADs are provided. Then, it highlights recent state of the art technologies and applications of PADs for various biomarker detection published during 2018 to May 2019. Applications for tumor marker, cardiac and coronary heath disease, and metabolic disorder biomarker detection are tabulated and selected examples are described in details. Finally, it discusses the current challenges, advances, and novel applications of PADs. The review would be valuable for healthcare workers using PADs as cost-effective point-of-care devices for biomarker detection and for researchers working on future developments of the devices.

Replacing synthetic polymers with renewable alternatives is a critical challenge for the packaging industry. This research investigated the use of leaf-based proteins as a sustainable co-binder in the coating formulations for paper-based packaging and other applications. Protein isolates from tobacco leaf and alfalfa concentrates were characterized using the Pierce protein assay, Kjeldahl nitrogen, and gel electrophoresis. The proteins were tested as co-binders in a typical latex-based paper coating formulation. The rheology and water retention properties of the wet coating and the surface, optical, structural, and strength properties of coated papers were measured. The coating performance was affected by the purity, solubility, and molecular weight of the tobacco protein and exhibited a shear-thinning behavior with lower water retention than soy protein. Analysis by scanning electron microscopy and time of flight secondary ion mass spectroscopy on the dried coating layer containing tobacco protein showed enhanced porosity (advantageous for package glueability) relative to the control latex coating. The tobacco protein offers adequate coverage and coating pigment distribution, indicating that this protein can be a suitable option in coatings for packaging applications.

This paper describes enzyme-linked immunosorbent assays (ELISAs) utilizing microfluidic thread/paper-based analytical devices (μTPAD), microfluidic fabric-based analytical devices (μFAD), and microfluidic thread-based analytical devices (μTAD). Here, the quantitative detection of biotinylated goat anti-mouse IgG (system one) and rabbit IgG (system two) antibodies via colorimetric analysis is detailed. In both systems, antibody is spotted on the detection site and subjected to a series of washes, addition of streptavidin-alkaline phosphatase (Strep-ALP) (system 1) or alkaline phosphatase (ALP)-conjugated secondary antibody (system 2), and colorimetric substrate. The devices are scanned and analyzed yielding a correlation between inverse yellow (or purple) intensity. For system one, a linear range of detection at low concentrations of streptavidin-alkaline phosphatase (Strep-ALP) was observed befire the enzyme reached a Vmax . At higher concentrations of Strep-ALP, saturation is achieved for both the μTPAD and μFAD devices. For system two, the IC50 values obtained for the non-trifurcated and trifurcated μTADs were determined to be 180.2 fmol/zone and 133.8 fmol/zone, respectively. The IC50 value was demonstrated to be 1034 fmol/zone and 208.6 fmol/zone for the μTPADs and μFADs, respectively. For all devices the lowest concentration of Strep-ALP or rabbit IgG used in the assay was 3.75 × 10-4 mg/mL and 0.7 fmol/zone, respectively. The development of this technology should further facilitate the use of these platforms for ELISA to detect and quantitate antibodies.

Fabric-based microfluidic fuel cells (MFCs) serve as a novel, cost-efficient alternative to traditional FCs and batteries, since fluids naturally travel across fabric via capillary action, eliminating the need for an external pump and lowering production and operation costs. Building on previous research with Y-shaped paper-based MFCs, fabric-based MFCs mitigate fragility and durability issues caused by long periods of fuel immersion. In this study, we describe a microfluidic fabric-based direct formate fuel cell, with 5 M potassium formate and 30% hydrogen peroxide as the anode fuel and cathode oxidant, respectively. Using a two-strip, stacked design, the optimized parameters include the type of encasement, the barrier, and the fabric type. Surface contact of the fabric and laminate sheet expedited flow and respective chemical reactions. The maximum current (22.83 mA/cm2 ) and power (4.40 mW/cm2 ) densities achieved with a 65% cotton/35% polyester blend material are a respective 8.7% and 32% higher than previous studies with Y-shaped paper-based MFCs. In series configuration, the MFCs generate sufficient energy to power a handheld calculator, a thermometer, and a spectrum of light-emitting diodes.

This work reports an investigation for the analysis of different paper samples using CE with laser-induced detection. Papers from four different manufactures (white-copy paper) and four different paper sources (white and recycled-copy papers, adhesive yellow paper notes and restaurant serviettes) were pulverized by scratching with a surgical scalpel prior to their derivatization with a fluorescent labeling agent, 8-aminopyrene-1,3,6-trisulfonic acid. Methodological conditions were evaluated, specifically the derivatization conditions with the aim to achieve the best S/N signals and the separation conditions in order to obtain optimum values of sensitivity and reproducibility. The best conditions, in terms of fastest, and easiest sample preparation procedure, minimal sample consumption, as well as the use of the simplest and fastest CE-procedure for obtaining the best analytical parameters, were applied to the analysis of the different paper samples. The registered electropherograms were pretreated (normalized and aligned) and subjected to multivariate analysis (principal component analysis). A successful discrimination among paper samples without entanglements was achieved. To the best of our knowledge, this work presents the first approach to achieve a successful differentiation among visually similar white-copy paper samples produced by different manufactures and paper from different paper sources through their direct analysis by CE-LIF and subsequent comparative study of the complete cellulose electropherogram by chemometric tools.

An exercise using simple paper strips to illustrate protein helical and sheet secondary structures is presented. Drawing on the rich historical context of the use of physical models in protein biochemistry by early practitioners, in particular Linus Pauling, the purpose of this activity is to cultivate in students a hands-on, intuitive sense of protein secondary structure and to complement the common computer-based structural portrayals often used in teaching biochemistry. As students fold these paper strips into model secondary structures, they will better grasp how intramolecular hydrogen bonds form in the folding of a polypeptide into secondary structure, and how these hydrogen bonds direct the overall shape of helical and sheet structures, including the handedness of the α-helix and the difference between right- and the left-handed twist. BIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION Vol. 39, No. 3, pp. 221–225, 2011.

A kinetic model was developed to predict batch simultaneous saccharification and co-fermentation (SSCF) of paper sludge by the xylose-utilizing yeast Saccharomyces cerevisiae RWB222 and the commercial cellulase preparation Spezyme CP. The model accounts for cellulose and xylan enzymatic hydrolysis and competitive uptake of glucose and xylose. Experimental results show that glucan and xylan enzymatic hydrolysis are highly correlated, and that the low concentrations of xylose encountered during SSCF do not have a significant inhibitory effect on enzymatic hydrolysis. Ethanol is found to not only inhibit the specific growth rate, but also to accelerate cell death. Glucose and xylose uptake rates were found to be competitively inhibitory, but this did not have a large impact during SSCF because the sugar concentrations are low. The model was used to evaluate which constants had the greatest impact on ethanol titer for a fixed substrate loading, enzyme loading, and fermentation time. The cellulose adsorption capacity and cellulose hydrolysis rate constants were found to have the greatest impact among enzymatic hydrolysis related constants, and ethanol yield and maximum ethanol tolerance had the greatest impact among fermentation related constants.

The simultaneous saccharification and co-fermentation (SSCF) kinetic model described in the companion paper can predict batch and fed batch fermentations well at solids concentrations up to 62.4 g/L cellulose paper sludge but not in batch fermentation at 82.0 g/L cellulose paper sludge. Four hypotheses for the discrepancy between observation and model prediction at high solids concentration were examined: ethanol inhibition, enzyme deactivation, inhibition by non-metabolizable compounds present in paper sludge, and mass transfer limitation. The results show that mass transfer limitation was responsible for the discrepancy between model and experimental data. The model can predict the value of high paper sludge SSCF in the fermentation period with no mass transfer limitation. The model predicted that maximum ethanol production of fed-batch fermentation was achieved when it was run as close to batch mode as possible with the initial solids loading below the mass transfer limitation threshold. A method for measuring final enzyme activity at the end of fermentation was also developed in this study.

Developing enzyme cocktails for cellulosic biomass hydrolysis complementary to current cellulase systems is a critical step needed for economically viable biofuels production. Recent genomic analysis indicates that some plant pathogenic fungi are likely a largely untapped resource in which to prospect for novel hydrolytic enzymes for biomass conversion. In order to develop high throughput screening assays for enzyme bioprospecting, a standardized microplate assay was developed for rapid analysis of polysaccharide hydrolysis by fungal extracts, incorporating biomass substrates. Fungi were grown for 10 days on cellulose- or switchgrass-containing media to produce enzyme extracts for analysis. Reducing sugar released from filter paper, Avicel, corn stalk, switchgrass, carboxymethylcellulose, and arabinoxylan was quantified using a miniaturized colorimetric assay based on 3,5-dinitrosalicylic acid. Significant interactions were identified among fungal species, growth media composition, assay substrate, and temperature. Within a small sampling of plant pathogenic fungi, some extracts had crude activities comparable to or greater than T. reesei, particularly when assayed at lower temperatures and on biomass substrates. This microplate assay system should prove useful for high-throughput bioprospecting for new sources of novel enzymes for biofuel production.

HLA-D/DR alleles as defined by cellular and serological typing can also be identified by biochemical methods. The Southern blot technique provides an additional typing facility which can be applied to DNA obtained from any source of nucleated cells. The polymorphism revealed by Southern blot analyses, the so-called restriction fragment length polymorphism (RFLP), depends upon the restriction enzyme and cDNA probes used. To identify HLA-DR specificities a protocol was developed based on the use of the results of southern blot analyses with several restriction enzymes and cDNA probes within a panel of HLA-D/DR homozygous cells representing the DR1 to DRw8 alleles. First, hybridizations with the 3' untranslated sequence of the DR beta cDNA probe, after digestion of the DNA with PvuII (PvuII-DR beta 3') allows the selective identification of DR1, DR2 and DRw8; DR3, DR5 and DRw6 are found as one group as well as DR4 and DR7 as another. Second, TaqI-DQ alpha hybridization allows the splitting of DRw6-Dw18, DRw6-Dw19 and DRw6-Dw9 from the DR3, DR5 and DRw6 group. The other alleles DR3, DR4, DR5, DRw6-Dw16 and DR7 are revealed by dehybridization and rehybridization of the blot with a DR beta cDNA probe. This protocol was used to test whether in a panel of 30 randomly chosen individuals the HLA-DR typing could be performed. The results were highly concordant to the serotyping. Furthermore by adding the Pst-DR beta and TaqI-DQ alpha RFLPs, most of the MLC defined Dw specificities could also be identified. An overview of the specific fragments described here has been summarized in matrices which can be used as references for DNA-typing in computerized analyses.

The 1- to 2-h illumination of xylanase with visible polarized light (PL) prior to the action of that enzyme upon beechwood xylan significantly increased its activity. The activity only negligibly decreased on 3 months storage. The hydrolysis of xylan proceeded in three well-distinguished stages. In the first and fastest stage the effect of illumination was only slightly positive. The effect of the stimulation was noted in the second, slower stage. Enzyme stimulated with PL, preferably by means of the 2-h illumination, performed better than enzyme stimulated with nonpolarized light and non-stimulated enzyme. In the last, the slowest stage, the rates of the reaction were nearly the same using either stimulated or non-stimulated enzyme.

The acute toxicity of fatty acids (C14 to C18) commonly found in wood was determined by the standard algal growth inhibition test using the freshwater green alga Selenastrum capricornutum. Toxicity, quantified as IC50 values, varied depending on the number of total carbons and double bonds. Of the tested acids, oleic (cis-9-octadecenoic) acid showed the highest toxicity (72-h IC50 = 0.47 mg/L) to the alga, and triolein, a triglyceride of oleic acid, showed no apparent toxicity. Further examination of a series of C18:1 acids with a double bond at the 6, 11, or 12 position revealed that both double-bond position and cis or trans configuration affected toxicity. The 72-h IC50 data for these fatty acids and related compounds seemed to correlate well with the melting point (mp), showing two separate linear relationships: at mp35 degrees C toxicity increased with increasing melting point, and at mp40 degrees C toxicity decreased with melting point.

Three enzyme preparations (crude cellulase, laccase, and proteinase) were evaluated for their potential to improve the papermaking properties of mechanical pulp. After treating a long fibre-rich fraction of the pulp with enzyme, the fibres were recombined with untreated fines for handsheet making and testing. None of the enzymes altered the retention of fines or the consolidation of the furnish mix during handsheet formation. All three enzymes increased tensile stiffness index, which is a measure of the initial resistance of the handsheets to strain. Only the laccase preparation, an enzyme that modifies pulp lignin, consistently increased fibre bonding to enhance other strength properties of the handsheets.

To develop an effective hybrid bioartificial liver (BAL) device, the material of the scaffold is very important to support hepatocytes that have both growth ability and hepatic differentiated functions. In this study we used paper (Kimwipe, Kimberly-Clark Corp., Roswell, GA, U.S.A.) as a scaffold. Primary hepatocytes isolated from a normal adult rat liver could proliferate on the paper. The secretion of albumin into culture medium by the cells on the paper increased with time in culture and, compared to the cells on dishes, the amount of 48 h albumin secretion at Day 10 was two times larger. Perpendicular sections of hepatocytes on the paper revealed that the cells fell into cavities made by intersecting fibers, piled up, and formed three to four layers. The piled-up cells changed their shape from flat to cuboidal and enlarged their cytoplasm, which was rich in organelles such as mitochondria and peroxisomes with a nucleoid. In addition, they formed bile canalicular structures between the cells. Their morphological appearance was similar to in vivo hepatocytes. Paper (Kimwipe) may be a good candidate as a scaffold to make a BAL device.

As part of a study on the microbiology of chlorate reduction, several new dissimilatory chlorate-reducing bacteria were isolated from a broad diversity of environments. One of these, strain CKB, was selected for a more complete characterization. Strain CKB was enriched and isolated from paper mill waste with acetate as the sole electron donor and chlorate as the sole electron acceptor. Strain CKB is a completely oxidizing, non-fermentative, Gram-negative, facultative anaerobe. Cells of strain CKB are 0.5 x 2 microm and are highly motile, with a single polar flagellum. In addition to acetate, strain CKB can use propionate, butyrate, lactate, succinate, fumarate, malate or yeast extract as electron donors, with chlorate as the sole electron acceptor. Strain CKB can also couple chlorate reduction to the oxidation of ferrous iron, sulphide, or the reduced form of the humic substances analogue 2,6-anthrahydroquinone disulphonate. Fe(II) is oxidized to insoluble amorphous Fe(II) oxide, whereas sulphide is oxidized to elemental sulphur. Growth is not associated with this metabolism, even when small quantities of acetate are added as a potential carbon source. In addition to chlorate, strain CKB can also couple acetate oxidation to the reduction of oxygen or perchlorate. Chlorate is completely reduced to chloride. Strain CKB has an optimum temperature of 35 degrees C, a pH optimum of 7.5 and a salinity optimum of 1% NaCl. Strain CKB can grow in chlorate and perchlorate concentrations of 80 or 20 mM respectively. Under anaerobic conditions, strain CKB can dismutate chlorite into chloride and O2, and is only the second organism shown to be capable of this metabolism. Oxidized minus reduced spectra of whole-cell suspensions of strain CKB showed absorbance maxima at 423, 523 and 552nm, which are indicative of the presence of c-type cytochrome(s). Analysis of the complete sequence of the 16S rDNA indicates that strain CKB is a member of the beta subclass of the Proteobacteria. The phototroph Rhodocyclus tenuis is the closest known relative. When tested, strain CKB could not grow by phototrophy and did not contain bacteriochlorophyll. Phenotypically and phylogenetically, strain CKB differs from all other described bacteria and represents the type strain of a new genus and species.

Nonfluorescing protein bands can be detected by the fluorescence optics of the commercial gel electrophoresis apparatus with automated scanning of the migration path (HPGE-1000, LabIntelligence, Belmont CA), taking advantage of the decrease of emission from a fluorescent paper placed below the gel by the absorbance of proteins ("fluorescence reduction"). That decrease of fluorescence gives rise to an inverted protein peak. Nonfluorescent colorless proteins appear to reduce the intensity of light emitted from the fluorescent paper due to absorbance of incident and emitted light. When the absorbance spectrum only slightly overlaps with the excitation and emission spectra of the fluorescent paper, that reduction is weak, and detection sensitivity in that application is consequently only 1/30 of that of fluorescent proteins. By contrast, when the protein is colored so that its absorbance spectrum overlaps widely with the excitation and emission spectra of the fluorescent paper, the sensitivity of "fluorescence reduction" equals 1/4 to 1/5 of that obtained for fluorescent proteins. Bands detected by "fluorescence reduction" provide a quantitative measure of protein load and mobility. The area of the inverted bands is proportional to protein loads up to 16 microg/lane of the gel tray. A theory of "fluorescence reduction" is presented which accounts for the existence of a linear relationship between band area and load.

The commercial production of ethanol from cellulose by simultaneous saccharification and fermentation (SSF) is prevented in part by the high cost of fungal cellulase enzymes. Intermittent exposure of SSF processes to ultrasonic energy under selected conditions (5 FPU of cellulase/g of substrate; 15 min of exposure/240 min cycle during the latter half of SSF) was found to increase ethanol production from mixed waste office paper by approximately 20%, producing 36.6 g/L ethanol after 96 h (70% of the maximum theoretical yield). Without ultrasound, 10 FPU of cellulase/g of substrate was required to achieve similar results. Continuous exposure of the organism to ultrasonic energy was bacteriostatic and decreased ethanol production but may be useful for the controlling bacterial growth in other processes.

Proteins immobilized on insoluble carriers or immunoadsorbents are now widely used in many areas of biological research. They are especially efficient in studies of complex protein mixtures because immunoadsorbents are very useful for isolation of individual components for further investigation. Antibodies immobilized on paper retain their specific activity and can be applied as very sensitive measurement tool.

The purpose of this study was to assess the recovery of certain proteins from paper strips used for sampling of gingival crevicular fluid (GCF). During a period of 60 min, proteins were eluent in isotonic phosphate-buffered saline, pH 7.4, without detergent. This eluent did not lyse neutrophils applied to the strips, which might otherwise contribute intracellular substances-, e.g., elastase to the GCF samples. The proteins applied (MW 12-725 kD) were satisfactorily recovered (about 90%). However, free neutrophil elastase, unlike elastase complexed with alpha-2-macroglobulin, could not be recovered from the paper strips. Strips made of polyvinylidene difluoride (Durapore) were also tested and 31% of the applied pure elastase was recovered from them. A comparison of measurements made with a Periotron 6000 on Periopaper and Durapore strips, showed that the values obtained with Durapore were too low for accurate measurements of small volumes of fluid. On the whole, when the elution was made in isotonic buffers at neutral pH and without detergents, the recovery of most proteins was satisfactory and reproducible, and independent of MW in the tested range. However, the study also showed poor recovery of uncomplexed elastase.

The preparation of homogeneous ultrathin native polyacrylamide gels, using a basic as well as an acidic buffer system is described. The basic buffer system consists of Tris-HC1/Tris-glycine, the same buffer as in sodium dodecyl sulfate (SDS)-gel electrophoresis but without SDS. The acidic system uses potassium acetate, pH 4.3, as gel buffer and beta-alanine, pH 4.6, acetic acid as electrolytes. The gels are covalently bound on glass plates. Binding of acidic gels requires a special pretreatment of glass plates. The whole procedure is simple and extraordinarily fast: 100-120 min from the start of gel preparation to the end of electrophoresis. Coomassie staining is done in 40 min and silver staining in 90 min. The native gels are excellently suited for diffusion blotting. Further attractive properties of these gels are easy handling, simple drying and dimensional stability.

Wild female and male white sucker (Catostomus commersoni) inhabiting an area receiving pulp mill effluent had reduced hepatic levels of retinol, didehydroretinol, retinyl esters, and didehydroretinyl esters, while vitamin E levels were unaffected. This disruption of the retinoid system led us to test methanol and dichloromethane extracts from the effluent of 11 pulp mills from across Canada for their ability to bind to rainbow trout (Oncorhynchus mykiss) retinoic acid receptors (RARs) from the gill and retinoid X receptors (RXRs) from the liver. Concentrated extracts of the final effluent from 6 of the 11 pulp mills were able to displace greater than 25% of the receptor-bound [3H]all-trans retinoic acid (RA) or [3H]9-cis RA from trout RARs and RXRs, respectively. The ability of the extracts to displace retinoic acid did not appear to be linked to the pulping or treatment processes. Moreover, extracts with the greatest activity came from thermomechanical mills, suggesting the compounds may originate from the wood furnish. In addition, extracts prepared from wood furnish (wood chips: white spruce [50%], lodgepole pine [47%], and balsam fir [3%]) from one mill were able to displace [3H]RA from the RARs and RXRs. The 4-hydroxy RA, a metabolite of RA that has been shown to be generated in greater quantities in fish exposed to P450-inducing xenobiotics, was able to displace [3H]all-trans RA from trout RARs as effectively as unlabeled all-trans RA. These results suggest that pulp mill effluent may impact the retinoid system of fish at multiple sites, either by decreasing hepatic retinoid stores or through contributing additional ligands (from the wood furnish) that can bind to RA receptors.

We have examined the expression on a panel of 22 human kidneys of polymorphic B-cell determinants recognized by mouse monoclonal antibodies. Monoclonal antibodies from a mouse immunized with an antigenic preparation from a DR4 positive B-cell line reacted preferentially with kidneys from DR4 positive donors (p less than 0.005), and the pattern of reactivity with kidney tissues was similar to that of antibodies to monomorphic determinants of class II. However, these antibodies did not show clear specificity for DR4 on lymphocytes in standard serological analyses. These results provide evidence for the expression of polymorphic class II determinants on human kidneys. Reasons for the differences in the apparent specificities of the monoclonal antibodies when tested on kidney sections and lymphocytes are discussed.

Endoglucanase CenA of Cellulomonas fimi comprises an N-terminal cellulose-binding domain and a C-terminal catalytic domain joined together by a sequence of 23 proline and threonine residues (the Pro-Thr box). The domains function independently when separated by proteolysis. TnphoA has been used to generate cenA′-′phoA fusions. CenA′-′PhoA fusion polypeptides which contain the entire cellulose-binding domain of CenA bind to cellulose, allowing their purification from periplasmic extracts in a single, facile step. This result has implications for purification or immobilisation of chimeric proteins on a cheap cellulose matrix.

Three previously defined antigenic subsets of C3 (C3(S), C3(D) alpha and C3(D)beta) clearly distinguish native C3 from C3 that has been denatured in the presence of sodium dodecyl sulphate (C3-SDS): C3-SDS expresses antigens of all 3 subsets, while native C3 only expressed C3(S) antigens. The radioimmunometric assessment performed in this study revealed that 90% of the C3(S) and C3(D)alpha and 70% of the C3(D)beta antigens demonstrable in C3-SDS also was expressed by physiologically bound C3b on sheep erythrocytes. The antigenic properties of bound C3b were not shared by soluble C3b, which (like soluble C3) only expressed C3(S) antigens, nor did soluble C3b 'spontaneously' released from complement-treated target cells express antigens other than C3(S). We therefore conclude that the expression of C3(D) antigens, under physiological conditions of activation, reflects a conformational modification unique for bound C3b and occurring to an extent comparable to that of C3 in the presence of a strong denaturant. Additional studies further revealed that the antigenic profile of bound C3b is determined by a heterogeneous population of molecules differing in their relative expression of the C3(S), C3(D)alpha and C3(D)beta subsets.

When soluble proteins in cytosolic fractions of rat soleus muscles are 32P-phosphorylated in vitro by an ATP:protein phosphotransferase reaction, the major substrate is a 56-kilodalton (56K) protein. As we have also reported previously, the onset and development of increased 32P-phosphorylation of this 56K protein, which are observed after the soleus is denervated, temporally correlate with the denervation period and length of the distal nerve stump [Held et al, 1983]. Conclusive evidence which identifies this neuroregulated muscle protein as the regulatory subunit of cyclic AMP-dependent protein kinase type II (R-II) is presented in this paper. The 56K soleus protein and purified bovine heart R-II were 32P-phosphorylated and subjected to limited proteolysis with bovine pancreas trypsin. After resolution of the generated 32P-phosphopeptides by SDS slab PAGE and visualization by autoradiography, no tryptic products were observed from the 56K soleus protein which were not also produced by proteolysis of the purified R-II. These tryptic phosphopeptides included 39, 16.5, and 12K fragments which retained the autophosphorylation site of R-II. After denervation, the 32P-phosphorylation of the 56K soleus protein and of the 39K tryptic peptide product were comparably increased. The identification of the neuroregulated 56K soleus protein as R-II was also confirmed by Western blotting with a specific anti-R-II sera. Taken together, our results demonstrate that the previously observed neuroregulation of the 32P-phosphorylation of the 56K soleus protein is identifiable with some alteration which affects the intramolecular 32P-autophosphorylation of R-II.

Male rats were fed 100 nM dichlorodiphenyltrichloroethane-14C in oil by gastric tube. Recovery of dichlorodiphenyltrichloroethane-14C in thoracic duct lymph was 60% in 12 hr. Lymph dichlorodiphenyltrichloroethane-14C (97%) occurred in lipoproteins of d

Several groups have attempted to identify the EGF receptor by a variety of biochemical techniques. Das et al. [l] identified the EGF receptor on 3T3 cells as a radioactive band of M, = 190 000 by photoaffmity labeling with a ‘251-EGF probe; Hock et al. [2] identified the EGF receptor in human placenta as a doublet of 160 000 and 180 000 M, by covalent cross-linking to ‘251-labeled EGF. The EGF receptor on A-43 1 cells was identified [3] as a radioactive band of M, 175 000 by ‘direct labeling’, which is accomplished by incubation of cells with 1251-EGF. Cohen and coworkers [4] have partially purified the EGF receptor of A-43 1 cells by affinity chromatography and analyzed the denatured proteins of such preparations by SDS-polyacrylamide gel electrophoresis. They found numerous protein bands in the affinity-purified preparations and suggested that a major protein band of M, 150 000 is the receptor for EGF in A-431 cells. More recently, Hunter and Cooper [S] isolated the EGF receptor from 32P-labeled A-43 1 cells by immunoprecipitation with an antiserum raised against crude plasma membrane from A-431 cells. They have estimated the M, of the EGF receptor present in the immunoprecipitates from A-43 1 cells as 155 000. The interaction of EGF with its membrane receptor, present in both crude plasma membranes from A-431 cells and in affinity-purified prepara

1. A low-molecular-weight and a high-molecular-weight 1,4beta-glucan glucanohydrolase (Cx enzyme) have been isolated from a commercial cellulase preparation derived from culture filtrates of the fungus Trichoderma viride. 2. The purification method for the isolation of the low-molecular-weight enzyme is a three-step procedure including chromatography on Bio-Gel P-10, chromatography on a dipolar adsorbent (arginine-Sepharose 6 B) and isoelectric focusing. 3. The starting material for the isolation of the high-molecular-weight enzyme was pre-fractionated by chromatography on Bio-Gel P-10, by DEAE-Sephadex chromatography and by SE-Sephadex chromatography as described previously by us. Further fractionation of this material was achieved by affinity chromatography and repeated isoelectric focusing. 4. Free zone electrophoresis of the low-molecular-weight enzyme indicated a homogeneous protein. The high-molecular-weight enzyme was homogenous in sedimentation equilibrium analysis. 5. The molecular weights of the enzymes were 12 500 and 50 000 +/- 2000 respectively. The former value was determined by chromatography on a calibrated column of Bio-Gel P-100 and the latter value by sedimentation equilibrium analysis. 6. The low-molecular-weight enzyme was isoelectric at pH 4.60 (10 degrees C) and contained 21% carbohydrate. The corresponding values for the high-molecular-weight enzyme were pH 3.39 and 12%. 7. Both enzymes were active in releasing free fibers from filter-paper. The low-molecular-weight enzyme was estimated to be about twice as effective as the high-molecular-weight enzyme in this regard.

The active thiol group of lombricine kinase from Lumbricus terrestris muscle was labelled with N-ethyl-[1-14C]maleimide. The resulting inactivated N-ethyl-[1-14C]succinimido enzyme was then subjected to tryptic hydrolysis. The peptide containing the labelled essential thiol group was isolated and found to contain: Leu-Gly-Tyr-Ile-Thr-[14C]Cys-Pro-Gly-Ser-Asn-Leu-Gly-Thr-Leu-Arg. The amino acid sequence around this thiol group was very similar with that of homologous ATP: guanidine phosphotransferases previously studied, arginine kinase from Homarus vulgaris muscle, creatine kinases from ox brain and ox muscle and from rabbit muscle. In addition among the other enzymes of this group, lombricine kinase is of special interest since it is the only dimeric enzyme of molecular weight ≃ 80000 which possesses only one essential thiol group and one nucleotide binding site per two subunits.

1 A new diagonal electrophoretic technique for determining the order of the lysine-containing tryptic peptides of a protein is described. The protein is converted into its trifluoracetyl derivative, digested enzymatically (or chemically), and the resulting peptides separated by paper electrophoresis. The paper is then treated with ammonia vapour, which re-exposes the ɛ-amino groups of the lysine residues, and submitted to a second electrophoresis at right angles to the first direction. Peptides containing lysine residues, together with the N-terminal peptide of the protein, are found to lie off a diagonal formed by all other peptides, whence they may be readily purified. A study of these peptides enables the order of the lysine-containing tryptic peptides in the protein to be deduced. 2 The technique has been successfully tested with insulin. 3 When the method was applied to porcine pepsin, the four tryptic peptides isolated were easily ordered and the carboxyl-terminal sequence of the protein shown to be Arg-Gln-Tyr-Tyr-Thr-Val-Phe-Asp-Arg-Ala-Asn-Asn-Lys-Val-Gly-Leu-Ala-Pro-Val-Ala. The three basic amino acid residues in the molecule are thus found clustering towards the C-terminus of the polypeptide chain. 4 A common ancestral gene for porcine pepsin and bovine (calf) rennin is suggested by the close homology between the C-terminal sequences of the two proteins.

Starting from 2-thiodeoxythymidine, the mono- and triphosphate of the nucleoside analog were prepared by enzymatic phosphorylation with thymidine kinase and thymidylate kinase, respectively. 2-Thiodeoxythymidine triphosphate was also obtained by chemical pyrophosphorylation of the monophosphate. The analog nucleoside-triphosphate is a substrate for Escherichia coli DNA polymerase. A copolymer consisting of alternating deoxyadenosine- and 2-thiodeoxythymidine nucleotides is synthesized extensively by the enzyme. The polymer was characterized by nearest neighbour analysis, sedimentation and buoyant density in the analytical ultracentrifuge, ultraviolet spectra and stability against thermal and alkaline denaturation.

From the lipopolysaccharides of two Escherichia coli strains (serotypes O 7:K1:(L):H− and O10:K5 (L):H4) two unusual amino sugars were isolated. By sequential oxidation with periodate and hypoiodite, followed by identification of the reaction products, the aminosugars were characterized as 4-amino-4,6-dideoxy-d-hexoses. The chromatographic properties of the amino sugars and their N-acetyl derivatives were compared with those of authentic samples of free and N-acetylated 4-amino-4,6-dideoxy-d-glucose and 4-amino-4,6-dideoxy-d-galactose. On the basis of the results obtained the unknown amino sugars were tentatively identified as 4-amino-4,6-dideoxy-d-glucose (from the lipopoly-saccharide of E. coli O7:K1:(L):H−) and 4-amino-4,6-dideoxy-d-galactose (from the lipopolysaccharide of E. coli O10:K5(L):H4). This is the first time that 4-amino-4,6-dideoxy-d-galactose is described as a constituent of a polysaccharide.

Mast cell granules, obtained from sonically disintegrated rat peritioneal cells and suspended in sucrose, 0.34 M, were found to release histamine in the presence of bee venom. Three venom fractions were obtained by gel filtration; two of these had strong histamine releasing capacity, namely “F I” containing phosphatidase A, and “F III” with direct hemolytic properties. The third fraction, “F II”, which releases histamine from intact mast cells, had little effect on granules. Various possibilities for the release mechanism, namely phosphatidase activity, surface activity, and cation exchange, are discussed.