Xerographic paper as a transfer medium for Western blots: Quantification of bovine αS1-casein by Western blot

We have found that xerographic paper (regular photocopy or laser printer paper) can be used as a transfer medium for protein blots for the immunodetection of low concentrations (a few nanograms) of bovine αS1-casein. With paper blotting, we could detect the protein with three times more sensitivity than with polyvinylidene difluoride. Blotting was achieved by transfer from sodium dodecyl sulfate-polyacrylamide gel electrophoresis to the methanol-wet paper. The blot was incubated with chicken anti-casein antibodies, sequentially peroxidase-labeled rabbit anti-chicken antibodies, and diaminobenzidine substrate. The blots were directly scanned and the pixel intensity of the band areas was integrated. An analysis of the scanned blots showed that the log of protein concentration was linearly related with the square root of an integrated pixel intensity (r² > 0.96). This linear relationship was observed in a wide range of protein concentration from 5 ng to 15 μg. The coefficients of variation were 12.4% for intraassay and 15.7% for interassay. This new analytical procedure can be applied to estimate the concentration of proteins by immunological blotting.     

Increase in constitutively active MEK1 species by introduction of MEK1 mutations identified in cancers

The kinase MEK1 is an essential component of the mitogen-activated protein kinase cascades. Somatic mutations that have been identified in the MEK1-coding gene generally enhance kinase activity. Consequently, MEK1 has attracted much interest as a target for cancer therapy to block the aberrant activity. By using Phos-tag affinity electrophoresis, we found that the introduction of mutations detected in certain sporadic cancers or in MEK-inhibitor-resistant cancer cells produced constitutively active MEK1 species containing phosphorylated Ser-218 and Ser-222 residues; it also enhanced the constitutive activity of the kinase. Phosphorylation profiling of the mutants in the presence of inhibitors of RAF/MEK demonstrated that several mutations conferred resistance to multiple inhibitors as a result of an increase in the quantity of active MEK1 species containing the two phosphorylated Ser-218 and Ser-222 residues. Phos-tag-based phosphorylation profiling of MEK1 can therefore provide clinical insights into characteristics of individual mutations in the MEK1-coding gene.     

A new DRB1 allele (DRB1 * 0811) identified in Native Americans

The nucleotide sequence data reported here have been submitted to the Genome Sequence Database and have been assigned the accession number L32810. The name DRB1 ^*0811 was officially assigned by the WHO Nomenclature Committee in March 1994. This follows the policy that, subject to the conditions stated in the most recent Nomenclature Report (Bodmer et al. 1992), names will be assigned to new sequences as they are identified. Lists of such new names will be published in the following WHO Nomenclature Report     

Novel aerobic benzene degrading microorganisms identified in three soils by stable isotope probing

The remediation of benzene contaminated groundwater often involves biodegradation and although the mechanisms of aerobic benzene biodegradation in laboratory cultures have been well studied, less is known about the microorganisms responsible for benzene degradation in mixed culture samples or at contaminated sites. To address this knowledge gap, DNA based stable isotope probing (SIP) was utilized to identify active benzene degraders in microcosms constructed with soil from three sources (a contaminated site and two agricultural sites). For this, replicate microcosms were amended with either labeled (¹³C) or unlabeled benzene and the extracted DNA samples were ultracentrifuged, fractioned and subject to terminal restriction fragment length polymorphism (TRFLP). The dominant benzene degraders (responsible for ¹³C uptake) were determined by comparing relative abundance of TRFLP phylotypes in heavy fractions of labeled benzene (¹³C) amended samples to the controls (from unlabeled benzene amended samples). Two phylotypes (a Polaromonas sp. and an Acidobacterium) were the major benzene degraders in the microcosms constructed from the contaminated site soil, whereas one phylotype incorporated the majority of the benzene-derived ¹³C in each of the agricultural soils (“candidate” phylum TM7 and an unclassified Sphingomonadaceae).     

Sequential detection of antigens in Western blots with differently colored products

A reliable sequential application of peroxidase and alkaline phosphatase-conjugated second antibodies to detection of two different antigens in Western blots is described. Deposits of differently colored products formed allow identification of several antigens on a single blot.     

Structural analysis of T4 DNA helix destabilizing protein (gp32 I) crystal by electron microscopy

Low dose electron diffraction and imaging techniques have been applied to the study of the crystalline structure of gp32*I, a DNA helix destabilizing protein derived from bacteriophage T4 gene 32 protein. A quantitative analysis of intensities from electron diffraction patterns from tilted, multilayered gp32*I crystal has provided the unit cell thickness of the crystal. The three-dimensional phases indicate that the space group P2(1)2(1)2. By taking into account the unit cell volume and the solvent content in the crystal, it was deduced that there is one gp32*I molecule in each asymmetric unit. A projected density map of unstained, glucose-embedded gp32*I crystal was synthesized with amplitudes from electron diffraction intensities and phases from electron images with reflections out to 7.6 A. Because of the similarity in the scattering density between glucose and protein, this projected map cannot be interpreted with certainty. A low resolution three-dimensional reconstruction shows that the protein molecule is about 90 A long and about 20 A in diameter. Because the dimer is formed around a dyad axis, the protein molecules comprising it must be arranged head-to-head. This dimeric arrangement of the proteins in the unit cell may be implicated as one of the conformational states of this protein in solution.     

Regulation of the expression on mouse T lymphocytes of the epitope identified by monoclonal antibody 3A35

Monoclonal antibody 3A35 (MA 3A35) has previously been shown to be an activation marker of macrophages and T lymphocytes. It immunoprecipitated from macrophages a 200-kDa molecule belonging to the T200 family and from T cells a 85-kDa antigen. In the present work, the factors controlling the expression of the epitope identified by MA 3A35 on polyclonal activated T cells and T-cell clones, as well as the ability of 3A35 alone or together with complement to interfere with T-cell functions, were investigated. Corticoresistant thymocytes unreactive with MA 3A35 became fully reactive after 2 days of in vitro stimulation by PMA and IL-2 and the level of reactivity per cell declined to a low level thereafter. In helper and cytolytic T-cell clones, the expression of the epitope defined by MA 3A35 was also maximal soon after antigenic stimulation then declined. In helper-T-cell clones, the epitope remained detectable during the entire culture period, whereas in cytolytic clones its expression was markedly reduced at the end of the culture. The lineage of cytotoxic T lymphocytes (CTL) as studied in a bulk culture of spleen cells primed in vivo against a syngeneic tumor exhibited similar regulation by antigenic stimulation. The CTL precursors were resistant to lysis by MA 3A35 plus complement; after 3 days of culture with the stimulatory antigen, they became highly sensitive but their sensitivity then diminished and mature CTL were completely resistant. MA 3A35 plus complement also killed the activated T cells which responded to macrophage-presented antigens and were thought to be mainly Lyt-1+. Therefore, the epitope identified by MA 3A35 was expressed predominantly at an early stage of T-cell activation. At a late stage, it persisted almost exclusively on helper and Lyt-1+ cells. In addition, MA 3A35 plus complement lysed NK cells, AK cells, and their precursors present in normal spleen. In the absence of complement, MA 3A35 had no detectable effect on T-cell functions.     

Solution probing of metal ion binding by helix 27 from Escherichia coli 16S rRNA

Helix (H)27 from Escherichia coli 16S ribosomal (r)RNA is centrally located within the small (30S) ribosomal subunit, immediately adjacent to the decoding center. Bacterial 30S subunit crystal structures depicting Mg(2+) binding sites resolve two magnesium ions within the vicinity of H27: one in the major groove of the G886-U911 wobble pair, and one within the GCAA tetraloop. Binding of such metal cations is generally thought to be crucial for RNA folding and function. To ask how metal ion-RNA interactions in crystals compare with those in solution, we have characterized, using solution NMR spectroscopy, Tb(3+) footprinting and time-resolved fluorescence resonance energy transfer (tr-FRET), location, and modes of metal ion binding in an isolated H27. NMR and Tb(3+) footprinting data indicate that solution secondary structure and Mg(2+) binding are generally consistent with the ribosomal crystal structures. However, our analyses also suggest that H27 is dynamic in solution and that metal ions localize within the narrow major groove formed by the juxtaposition of the loop E motif with the tandem G894-U905 and G895-U904 wobble pairs. In addition, tr-FRET studies provide evidence that Mg(2+) uptake by the H27 construct results in a global lengthening of the helix. We propose that only a subset of H27-metal ion interactions has been captured in the crystal structures of the 30S ribosomal subunit, and that small-scale structural dynamics afforded by solution conditions may contribute to these differences. Our studies thus highlight an example for differences between RNA-metal ion interactions observed in solution and in crystals.     

Inhibition of mouse myeloma DNA polymerase alpha by 5-triphosphates of 1-beta-D-arabinofuranosylthymine and 1-beta-D-arabinofuranosylcytosine

This is the first report dealing with the effect of 1-beta-D-arabinofuranosylthymine 5'-triphosphate (araTTP), synthesized by a new method, on eukaryotic DNA polymerase [EC 2.7.7.7]. AraTTP was tested for the inhibition of DNA synthesis in vitro using highly purified mouse myeloma DNA polymerase alpha in comparison with 1-beta-D-arabinofuranosylcytosine 5'-triphosphate (araCTP). AraTTP was found to inhibit competitively the incorporation of [3H]dTTP into DNA and non-competitively the incorporation of [3H]dCTP, while the mode of the inhibition by araCTP was non-competitive with respect to dTTP and competitive with respect to dCTP. Neither araTTP nor araCTP was utilized as a substrate in place of dTTP or dCTP in DNA synthesis by DNA polymerase alpha.     

Acrosomal constituents identified with a monoclonal antibody are modified during late spermiogenesis in the mouse

Monoclonal antibody 1D4, a mouse immunoglobulin M raised against CD-1 mouse spermatogenic cell membranes, recognizes acrosomal constituents in the mouse, rabbit, and guinea pig. In the mouse, acrosomes of round and condensing spermatids were labeled with 1D4 by indirect immunofluorescence on isolated cells and by immunohistochemistry on paraffin sections. During the terminal steps of spermiogenesis, however, acrosomal labeling in mouse germ cells was lost. Little or no 1D4 immunoreactivity was detected by enzyme-linked immunosorbent assays in prepubertal testes, Sertoli cells, or several somatic tissues. To identify antigens recognized by 1D4, mouse spermatogenic cell proteins were separated by one- (1D) and two-dimensional (2D) polyacrylamide gel electrophoresis, transferred to nitrocellulose, and immunostained. Multiple antigens larger than 200,000 relative molecular weight (Mr) were resolved on 1D immunoblots from round and condensing spermatids isolated by sedimentation velocity at unit gravity. A smaller antigen (Mr 85,000 isoelectric point approximately 5.7) was also detected on 1D and 2D immunoblots of round spermatid proteins. These antigens can be labeled biosynthetically with [3H] glucosamine and immunoprecipitated, suggesting that they are a set of glycoconjugates that share a common epitope recognized by 1D4. This determinant is no longer detectable in late spermatids, indicating that biochemical modifications of acrosomal constituents occur during the terminal steps of germ cell differentiation.     

Induction of activator protein-1 through reactive oxygen species by crystalline silica in JB6 cells

We reported previously that freshly fractured silica (FFSi) induces activator protein-1 (AP-1) activation through extracellular signal-regulated protein kinases (ERKs) and p38 kinase pathways. In the present study, the biologic activities of FFSi and aged silica (ASi) were compared by measuring their effects on the AP-1 activation and phosphorylation of ERKs and p38 kinase. The roles of reactive oxygen species (ROS) in this silica-induced AP-1 activation were also investigated. We found that FFSi-induced AP-1 activation was four times higher than that of ASi in JB6 cells. FFSi also caused greater phosphorylation of ERKs and p38 kinase than ASi. FFSi generated more ROS than ASi when incubated with the cells as measured by electron spin resonance (ESR). Studies using ROS-sensitive dyes and oxygen consumption support the conclusion that ROS are generated by silica-treated cells. N-Acetylcysteine (an antioxidant) and polyvinyl pyridine-N-oxide (an agent that binds to Si-OH groups on silica surfaces) decreased AP-1 activation and phosphorylation of ERKs and p38 kinase. Catalase inhibited phosphorylation of ERKs and p38 kinase, as well as AP-1 activation induced by FFSi, suggesting the involvement of H(2)O(2) in the mechanism of silica-induced AP-1 activation. Sodium formate (an ( small middle dot)OH scavenger) had no influence on silica-induced MAPKs or AP-1 activation. Superoxide dismutase enhanced both AP-1 and MAPKs activation, indicating that H(2)O(2), but not O(2), may play a critical role in silica-induced AP-1 activation. These studies indicate that freshly ground silica is more biologically active than aged silica and that ROS, in particular H(2)O(2), play a significant role in silica-induced AP-1 activation.     

Anaerobic methyl tert-butyl ether-degrading microorganisms identified in wastewater treatment plant samples by stable isotope probing

Anaerobic methyl tert-butyl ether (MTBE) degradation potential was investigated in samples from a range of sources. From these 22 experimental variations, only one source (from wastewater treatment plant samples) exhibited MTBE degradation. These microcosms were methanogenic and were subjected to DNA-based stable isotope probing (SIP) targeted to both bacteria and archaea to identify the putative MTBE degraders. For this purpose, DNA was extracted at two time points, subjected to ultracentrifugation, fractioning, and terminal restriction fragment length polymorphism (TRFLP). In addition, bacterial and archaeal 16S rRNA gene clone libraries were constructed. The SIP experiments indicated bacteria in the phyla Firmicutes (family Ruminococcaceae) and Alphaproteobacteria (genus Sphingopyxis) were the dominant MTBE degraders. Previous studies have suggested a role for Firmicutes in anaerobic MTBE degradation; however, the putative MTBE-degrading microorganism in the current study is a novel MTBE-degrading phylotype within this phylum. Two archaeal phylotypes (genera Methanosarcina and Methanocorpusculum) were also enriched in the heavy fractions, and these organisms may be responsible for minor amounts of MTBE degradation or for the uptake of metabolites released from the primary MTBE degraders. Currently, limited information exists on the microorganisms able to degrade MTBE under anaerobic conditions. This work represents the first application of DNA-based SIP to identify anaerobic MTBE-degrading microorganisms in laboratory microcosms and therefore provides a valuable set of data to definitively link identity with anaerobic MTBE degradation.     

Multiple reprobing of Western blots after inactivation of peroxidase activity by its substrate, hydrogen peroxide

Sequential detections of different proteins on Western blot save time and precious samples. The main problem concerning reprobing is that stripping buffers can unbind both the antibody and the tested antigen. An original reprobing method has been set up based on horseradish peroxidase (HRP) inhibition after enhanced chemiluminescence detection. Instead of removing previously fixed antibodies as common stripping buffers do, the HRP activity linked to the secondary antibody is irreversibly inhibited by excess of hydrogen peroxide. A 15-min incubation allows one to perform at least five different sequential detections without losing significant amounts of blotted proteins.     

Marginal zone macrophages in the mouse spleen identified by a monoclonal antibody. Anatomical correlation with a B cell subpopulation

The reactivity of a monoclonal antibody, ER-TR9, that demonstrates heterogeneity among mononuclear phagocytes is described. In the spleen, ER-TR9 exclusively reacts with a population of macrophages located in the marginal zone. ER-TR9 does not react with macrophage antigen 1-positive red pulp macrophages or any other types of splenic stromal cells. ER-TR9+ ve cells localize in anatomical proximity of a subpopulation of B cells, i.e., B cells that are immunoglobulin M positive and weakly positive to negative for immunoglobulin D. The possible significance of this particular interaction between both cell types during the immune response is discussed.     

Reticular fibroblasts in peripheral lymphoid organs identified by a monoclonal antibody

We have produced a panel of monoclonal antibodies directed against nonlymphoid cells in central and peripheral lymphoid organs. In this paper we present the reactivity of one of these antibodies, ER-TR7. This antibody detects reticular fibroblasts, which constitute the cellular framework of lymphoid and nonlymphoid organs and their products. In frozen sections of the spleen incubated with this antibody, the red pulp and white pulp are clearly delineated. Furthermore, the major white pulp compartments--the follicles and periarteriolar lymphoid sheath as well as the marginal zone--are recognized by their characteristic labeling patterns. In lymph nodes, the capsule, sinuses, follicles, paracortex, and medullary cords are clearly delineated. In the thymus and bone marrow no such specialized compartments were demonstrated. ER-TR7 reacts with an intracellular component of fibroblasts. Since ER-TR7 does not react with purified laminin, collagen types I-V, fibronectin, heparan sulfate proteoglycan, entactin, or nidogen, it detects a hitherto uncharacterized antigen. The possible role of the ER-TR7 positive reticular fibroblasts in the cellular organization of peripheral lymphoid organs will be discussed.     

A duplex approach for immunochemical staining and typing of proteins in Western blots

The qualitative and semiquantitative Western blotting technique enables the detection of separate proteins and the determination of subtypes and fragments by specific immunological reactions. Protein typing on immunoblots is restricted to antibody-specific determination, with the result of a specific banding pattern. For protein characterization, several antibodies that recognize different epitopes within the protein sequence are used. However, repeated or parallel gel runs are needed. Here we describe a sequential determination of prion proteins in healthy and pathological states that both consist of di-, mono-, and nonglycosylated isoforms using a single blot with two antibodies from two species that recognize one antigen with two epitopes. The band signals are visualized by using different chemiluminescent substrate reactions. This application can be used in the fields of diagnostics and public health to detect full-length and fragmented proteins and can also be used for characterization of overlaying proteins.