Properties of proteins in cancer procoagulant preparations that are detected by anti-tissue factor antibodies

Cancer procoagulant (CP) and tissue factor (TF; only in complex with Factor VIIa (FVIIa)) can activate FX to FXa. Controversy still exists whether or not CP is an entity different from TF, or whether CP activity is due to contamination of CP preparations with TF/FVIIa complex. We therefore looked for proteins in CP preparations that were detected by anti-TF antibodies and then sequenced these proteins. One- and two-dimensional gels of CP and TF were used to identify proteins immunoreactive to monoclonal anti-CP and anti-TF antibodies (Mabs). Those proteins in the CP preparation recognized by anti-TF antibodies were sequenced. Angiotensinogen precursor, alpha-1-antitrypsin precursor, and vitamin D-binding protein were identified along with one so far unidentified sequence; however, no TF-sequences were identified. Also, no proteins with the correct molecular weight for TF were identified using anti-TF antibodies. It seems possible that CP preparations contain proteins that have some epitopes similar to the epitopes recognized in TF by anti-TF Mab. However, these proteins do neither have the molecular weight nor the amino acid sequence of TF.     

Mechanisms of chromosome banding. II. Evidence that histones are not involved

C-and G-banding in mouse metaphase chromosomes is unaffected by exposure of the chromosomes to 0.2 N HCl for 4 h. Electrophoretic studies indicate that this is adequate to completely remove the histones from fixed and dried chromatin thus indicating that histones are not involved in C- or G-banding.     

Monoclonal antibodies that detect live salmonellae.

Nine immunoglobulin G and nine immunoglobulin M murine monoclonal antibody-producing hybridomas reactive with live Salmonella bacteria were obtained from several fusions of immune spleen cells and Sp2/0 myeloma cells. The antibodies were selected by the magnetic immunoluminescence assay. The monoclonal antibodies were reactive with serogroups A, B, C1, C2, D, E, and K and Salmonella choleraesuis subsp. diarizonae. Each monoclonal antibody proved to be reactive with a distinct serotype. Clinical isolates belonging to these Salmonella serogroups could be detected. Reactivity with non-Salmonella bacteria proved to be minor.     

Paraspeckles are subpopulation-specific nuclear bodies that are not essential in mice

Nuclei of higher organisms are well structured and have multiple, distinct nuclear compartments or nuclear bodies. Paraspeckles are recently identified mammal-specific nuclear bodies ubiquitously found in most cells cultured in vitro. To investigate the physiological role of paraspeckles, we examined the in vivo expression patterns of two long noncoding RNAs, NEAT1_1 and NEAT1_2, which are essential for the architectural integrity of nuclear bodies. Unexpectedly, these genes were only strongly expressed in a particular subpopulation of cells in adult mouse tissues, and prominent paraspeckle formation was observed only in the cells highly expressing NEAT1_2. To further investigate the cellular functions of paraspeckles, we created an animal model lacking NEAT1 by gene targeting. These knockout mice were viable and fertile under laboratory growth conditions, showing no apparent phenotypes except for the disappearance of paraspeckles. We propose that paraspeckles are nonessential, subpopulation-specific nuclear bodies formed secondary to particular environmental triggers.     

Monoclonal antibodies that detect live salmonellae.

Nine immunoglobulin G and nine immunoglobulin M murine monoclonal antibody-producing hybridomas reactive with live Salmonella bacteria were obtained from several fusions of immune spleen cells and Sp2/0 myeloma cells. The antibodies were selected by the magnetic immunoluminescence assay. The monoclonal antibodies were reactive with serogroups A, B, C1, C2, D, E, and K and Salmonella choleraesuis subsp. diarizonae. Each monoclonal antibody proved to be reactive with a distinct serotype. Clinical isolates belonging to these Salmonella serogroups could be detected. Reactivity with non-Salmonella bacteria proved to be minor.     

Evidence that Enzyme Polymorphisms are not Selectively Neutral

THE discovery of large amounts of electrophoretically-detectable genetic variation within natural populations has aroused considerable interest in the factors maintaining so much polymorphism. It has been proposed that these allozyme polymorphisms reflect the action of random processes^1–6, the polymorphic variation being of little or no selective significance. Alternative hypotheses have suggested that these polymorphisms may be maintained by a balance of selective forces^7–10.     

Mitochondrial DNA barcoding detects some species that are real, and some that are not

Mimicry and extensive geographical subspecies polymorphism combine to make species in the ithomiine butterfly genus Mechanitis (Lepidoptera; Nymphalidae) difficult to determine. We use mitochondrial DNA (mtDNA) barcoding, nuclear sequences and amplified fragment length polymorphism (AFLP) genotyping to investigate species limits in this genus. Although earlier biosystematic studies based on morphology described only four species, mtDNA barcoding revealed eight well-differentiated haplogroups, suggesting the presence of four new putative 'cryptic species'. However, AFLP markers supported only one of these four new 'cryptic species' as biologically meaningful. We demonstrate that in this genus, deep genetic divisions expected on the basis of mtDNA barcoding are not always reflected in the nuclear genome, and advocate the use of AFLP markers as a check when mtDNA barcoding gives unexpected results.     

Inferences about nested subsets structure when not all species are detected

Comparisons of species composition among isolated ecological communities of different size have often provided evidence that the species in communities with lower species richness form nested subsets of the species in larger communities. In the vast majority of studies, the question of nested subsets has been addressed using information on presence‐absence, where a “0” is interpreted as the absence of a given species from a given location. Most of the methodological discussion in earlier studies investigating nestedness concerns the approach to generation of model‐based matrices corresponding to the null hypothesis of a nonnested pattern. However, it is most likely that in many situations investigators cannot detect all the species present in the location sampled. The possibility that zeros in incidence matrices reflect nondetection rather than absence of species has not been considered in studies addressing nested subsets, even though the position of zeros in these matrices forms the basis of earlier inference methods. These sampling artifacts are likely to lead to erroneous conclusions about both variation over space in species richness, and the degree of similarity of the various locations. Here we propose an approach to investigation of nestedness, based on statistical inference methods explicitly incorporating species detection probability, that take into account the probabilistic nature of the sampling process. We use presence‐absence data collected under Pollock's robust capture‐recapture design, and resort to an estimator of species richness originally developed for closed populations to assess the proportion of species shared by different locations. We develop testable predictions corresponding to the null hypothesis of a nonnested pattern, and an alternative hypothesis of perfect nestedness. We also present an index for assessing the degree of nestedness of a system of ecological communities. We illustrate our approach using avian data from the North American Breeding Bird Survey collected in Florida Keys.     

Modified dsRNAs that are not processed by Dicer maintain potency and are incorporated into the RISC

Chemical modification of RNA duplexes can provide practical advantages for RNA interference (RNAi) triggering molecules including increased stability, safety and specificity. The impact of nucleotide modifications on Dicer processing, RISC loading and RNAi-mediated mRNA cleavage was investigated with duplexes ≥25 bp in length. It is known that dsRNAs ≥25 bp are processed by Dicer to create classic 19-bp siRNAs with 3′-end overhangs. We demonstrate that the presence of minimal modification configurations on longer RNA duplexes can block Dicer processing and result in the loading of the full-length guide strand into RISC with resultant mRNA cleavage at a defined site. These longer, modified duplexes can be highly potent gene silencers, with EC50s in the picomolar concentration range, demonstrating that Dicer processing is not required for incorporation into RISC or potent target silencing.     

Novel object exploration in mice: not all objects are created equal

Object exploration is an increasingly popular experimental paradigm in behavioral sciences. We have begun a series of studies with mice specifically looking at the parameters that influence behaviors in this test. The aim of the present study was to examine the effect of object type on performance in the object exploration test. More specifically, adult male C57BL/6J mice were trained and tested using objects that could be climbed (CLIMB) or with those that could only be touched (TOUCH). The results show that activity is affected by the presentation of objects, with object type interacting with some of these changes. C57 mice explored objects that can be climbed over significantly longer than objects that can only be touched and a more rapid habituation was observed using objects that could only be touched. Robust recognition memory was observed in both groups of mice, however mice in the CLIMB group exhibited a significantly greater discrimination index compared to mice in the TOUCH group. Taken together, these findings demonstrate that the selection of objects is of critical importance and it is recommended that special attention be given to the functional (affordant) properties of the objects to-be-used in future studies.     

Antibiotic-tolerant mutants of Streptococcus pneumoniae that are not deficient in autolytic activity.

Several mutants of Streptococcus pneumoniae were isolated that appeared tolerant, to varying extents, to the lytic and bactericidal effects of some antibiotics that inhibit peptidoglycan synthesis, but were not deficient in autolytic activity. The method used to select the mutants was based on the survival of tolerant mutants during treatment with either bacitracin, benzylpenicillin, D-cycloserine plus beta-chloro-D-alanine, or vancomycin. Most (60 to 80%) of the surviving isolates were found to be deficient in autolytic activity, and these were rejected. The smaller proportion that had wild-type sensitivity to deoxycholate-induced lysis was studied further with respect to tolerance to the other antibiotics used in the selection procedures. Two of these mutants (selected by treatment with benzylpenicillin) were tolerant to either benzylpenicillin or D-cycloserine plus beta-chloro-D-alanine, but were supersusceptible, in terms of initiation of lysis, to either bacitracin or vancomycin. The minimal inhibitory concentration values of several antibiotics for these two mutants were identical to those for the wild-type strain. Moreover, the interaction of radioactive benzylpenicillin with the penicillin-binding proteins, examined in whole organisms, also appeared the same as previously found for either wild-type or autolytic-deficient strains of S. pneumoniae.     

Staphylococcus aureus and Micrococcus luteus peptidoglycan transglycosylases that are not penicillin-binding proteins.

Major peptidoglycan transglycosylase activities, which synthesize uncross-linked peptidoglycan from lipid-linked precursors, were solubilized from the membranes of Staphylococcus aureus and Micrococcus luteus and were partially purified. The transglycosylase activities were separated from penicillin-binding proteins by solubilization and by purification steps. Therefore, we concluded that these activities were not activities of the penicillin-binding proteins, which are the presumptive peptidoglycan transpeptidases in these gram-positive cocci. Unlike Escherichia coli, in which the network structure of peptidoglycan is synthesized by multiple two-headed penicillin-binding proteins with both transpeptidase and transglycosylase activities, these gram-positive cocci have cell wall peptidoglycan which seems to be synthesized by penicillin-binding protein transpeptidases and a separate transglycosylase.     

Structure predictions of membrane proteins are not that bad

A simple generalization of the well-known hydrophobicity analysis is sufficient to predict membrane-spanning amphiphilic alpha-helices and beta-strands. The use of this method is illustrated for bacteriorhodopsin, OmpA protein and the photosynthetic reaction centre.