Autologous antibody response against the principal neutralizing domain of human immunodeficiency virus type 1 isolated from infected humans.

High titers of neutralizing antibodies in human immunodeficiency virus type 1 (HIV-1) infection are directed primarily against the third hypervariable domain (V3) of the virion envelope glycoprotein gp120. This region has been designated the principal neutralizing domain of HIV-1. Because the frequency and significance of autologous V3 antibodies in natural infection are not fully clarified, we have cloned, sequenced, and expressed the V3 domain from virus of HIV-1-infected patients to test the autologous and heterologous V3 antibody response. The resulting recombinant Escherichia coli V3 fusion proteins reacted strongly with both autologous and heterologous patient antibodies in Western blots. Thirty-one different V3 fragments were cloned from 24 hemophiliac patients with different immunological and clinical statuses. Antibody reactivity against the autologous V3 fusion proteins was detected in all serum samples except one; moreover, all serum samples contained antibody reactivity against a vast majority of heterologous fusion proteins despite significant amino acid variability in V3. The results suggest that V3 antibodies are highly prevalent; further, we find no association between the stage of the HIV-1 infection and the presence of V3 antibodies.     

Partial characterization of the carbohydrate units of rat intestinal sucrase--isomaltase.

Sucrase--isomaltase was purified from rat intestinal microvillus membranes after injection of D-[2-3H]mannose and L-[6-3H]fucose, using a column of monoclonal antibody-protein A-Sepharose. After Pronase digestion and gel filtration of the glycopeptides labelled from both precursors, a major part of the radioactivity was recovered in asparagine-linked complex oligosaccharides, and a smaller amount in partially alkali-labile high-molecular-weight glycopeptides. Only a small amount of [3H]mannose was found in endo-beta-N-acetylglucosaminidase H-sensitive high-mannose oligosaccharides.     

Imaging of anthrax intoxication in mice reveals shared and individual functions of surface receptors CMG-2 and TEM-8 in cellular toxin entry

Bacillus anthracis lethal toxin and edema toxin are binary toxins that consist of a common cell-binding moiety, protective antigen (PA), and the enzymatic moieties, lethal factor (LF) and edema factor (EF). PA binds to either of two receptors, capillary morphogenesis protein-2 (CMG-2) or tumor endothelial marker-8 (TEM-8), which triggers the binding and cytoplasmic translocation of LF and EF. However, the distribution of functional TEM-8 and CMG-2 receptors during anthrax toxin intoxication in animals has not been fully elucidated. Herein, we describe an assay to image anthrax toxin intoxication in animals, and we use it to visualize TEM-8- and CMG-2-dependent intoxication in mice. Specifically, we generated a chimeric protein consisting of the N-terminal domain of LF fused to a nuclear localization signal-tagged Cre recombinase (LFn-NLS-Cre). When PA and LFn-NLS-Cre were coadministered to transgenic mice expressing a red fluorescent protein in the absence of Cre and a green fluorescent protein in the presence of Cre, intoxication could be visualized at single-cell resolution by confocal microscopy or flow cytometry. Using this assay, we found that: (a) CMG-2 is critical for intoxication in the liver and heart, (b) TEM-8 is required for intoxication in the kidney and spleen, (c) CMG-2 and TEM-8 are redundant for intoxication of some organs, (d) combined loss of CMG-2 and TEM-8 completely abolishes intoxication, and (e) CMG-2 is the dominant receptor on leukocytes. The novel assay will be useful for basic and clinical/translational studies of Bacillus anthracis infection and for clinical development of reengineered toxin variants for cancer treatment.     

Biosystematic studies of Sorbus meinichii (Rosaceae) at Moster, S. Norway

Morphometric and cytological data were used to test the variation within the apomictic hybrid Sorbus meinichii from its type locality, in the Moster area, Bømlo in Sunnhordland, Norway. A lectotype is selected for the name. A total of 36 morphological characters were measured on leaves, stomata, flowers, pollen, fruits and seeds, from randomly selected S. meinichii individuals. In the field the material was classified into two groups, one including individuals of generally larger dimensions of leaves and fruits than the other. The preliminary classification was subsequently tested by a multivariate analysis of several characters, substantiating the existence of two morphs in the population at the type locality of S. meinichii , one corresponding to the selected lectotype. Assessment of the nature and taxonomic rank of these two morphs must await further studies. Cytological studies showed both forms of S. meinichii to be triploid, with 2n=51 chromosomes. The taxonomic separation of S. teodorii from S. meinichii on the basis of ploidy level is therefore not corroborated. Sorbus meinichii s. lat. was shown to be intermediate between its supposed parents, S. aucuparia and S. hybrida , in several leaf characters. All of the variation within the S. aucuparia × S. hybrida-complex in Fennoscandia is better included in one aggregate species Sorbus meinichii according to the present state of knowledge.     

Nuclear and mitochondrial DNA repair: similar pathways?

Mitochondrial DNA (mtDNA) alterations are implicated in a broad range of human diseases and alterations of the mitochondrial genome are assumed to be a result of its high susceptibility to oxidative damage and its limited DNA repair compared to nuclear DNA (nDNA). Characterization of DNA repair mechanisms has generally focused on these processes in nDNA but increasing interest and research effort have contributed to our knowledge of the mechanisms underlying DNA repair in mitochondria. In this review, we make comparisons between nDNA and mtDNA repair pathways and propose a model for how these pathways interact in mitochondria.     

Choline-binding protein D (CbpD) in Streptococcus pneumoniae is essential for competence-induced cell lysis

Streptococcus pneumoniae is an important human pathogen that is able to take up naked DNA from the environment by a quorum-sensing-regulated process called natural genetic transformation. This property enables members of this bacterial species to efficiently acquire new properties that may increase their ability to survive and multiply in the human host. We have previously reported that induction of the competent state in a liquid culture of Streptococcus pneumoniae triggers lysis of a subfraction of the bacterial population resulting in release of DNA. We have also proposed that such competence-induced DNA release is an integral part of natural genetic transformation that has evolved to increase the efficiency of gene transfer between pneumococci. In the present work, we have further elucidated the mechanism behind competence-induced cell lysis by identifying a putative murein hydrolase, choline-binding protein D (CbpD), as a key component of this process. By using real-time PCR to estimate the amount of extracellular DNA in competent relative to noncompetent cultures, we were able to show that competence-induced cell lysis and DNA release are strongly attenuated in a cbpD mutant. Ectopic expression of CbpD in the presence or absence of other competence proteins revealed that CbpD is essentially unable to cause cell lysis on its own but depends on at least one additional protein expressed during competence.     

Effects of Diverse Environmental Conditions on φLC3 Prophage Stability in Lactococcus lactis

The effects of various growth conditions on spontaneous LC3 prophage induction in Lactococcus lactis subsp. cremoris IMN-C1814 was analyzed with a half fraction of a 4⁴ factorial experimental design. The four factors included in the study were nutrient availability, acidity, osmolarity, and temperature, each applied at four levels. These environmental factors are related to the fermentation processes in the dairy industry, in which bacteriophage attacks on sensitive starter strains are a constant threat to successful fermentation processes. The frequency of spontaneous LC3 induction was determined by quantitative analyses of restored DNA attachment sites (attB) on the bacterial chromosomes in a population of lysogenic cells. Statistical analysis revealed that all four environmental factors tested affected LC3 prophage stability and that the environmental factors were involved in interactions (interactions exist when the effect of one factor depends on the level of another factor). The spontaneous LC3 induction frequency varied from 0.08 to 1.76%. In general, the induction frequency remained at the same rate or decreased when level 1 to 3 of the four environmental factors was applied. At level 4, which generally gave the least favorable growth conditions, the induction frequency was either unchanged, decreased, or increased, depending on the type of stress. It appeared that the spontaneous induction frequency was independent of the growth behavior of the host. It was the environmental growth conditions that were the decisive factor in induction frequency.     

Optical zymography for specific detection of urokinase plasminogen activator activity in biological samples

Zymography techniques are routinely used to quantify proteolytic activity. In the current study, we describe an optical zymographic procedure that specifically detects urokinase-type plasminogen activator (uPA) activity in biological samples. The method employs a synthetic polymeric uPA fluorescent probe, which is copolymerized in sodium dodecyl sulfate (SDS)-polyacrylamide gel. Following electrophoresis and renaturation, enzymatic digestions of the substrate in 50 mM of Tris buffer at pH 7.4 generates fluorescence emission at 695 nm. The enzymatic activities can be analyzed directly by conventional gel imaging systems with a detection limit of 40 pg. This protocol is fast (hours) and does not require staining and destaining steps. The procedure is independent of plasminogen and, therefore, can efficiently distinguish the active two-chain uPA from its proenzyme. Densitometry analysis demonstrated a highly correlative relationship (r2=0.999) between the amount of uPA (over the range of 0.1-8.0 ng) and the average intensity of the fluorescent band. We were able to directly measure uPA activities in different cancer cell lines. This newly developed technique could be expanded to nearly all proteases, including the ones that cannot be analyzed by traditional zymography.     

A high-resolution comparative map between pig chromosome 17 and human chromosomes 4, 8, and 20: identification of synteny breakpoints

We report on the construction of a high-resolution comparative map of porcine chromosome 17 (SSC17) focusing on evolutionary breakpoints with human chromosomes. The comparative map shows high homology with human chromosome 20 but suggests more limited homologies with other human chromosomes. SSC17 is of particular interest in studies of chromosomal organization due to the presence of QTLs that affect meat quality and carcass composition. A total of 158 pig ESTs available in databases or developed by the Sino-Danish Pig Genome Sequencing Consortium were mapped using the INRA-University of Minnesota porcine radiation hybrid panel. The high-resolution map was further anchored by fluorescence in situ hybridization. This study confirmed the extensive conservation between SSC17 and HSA20 and enabled the gene order to be determined. The homology of the SSC17 pericentromeric region was extended to other human chromosomes (HSA4, HSA8) and the chromosomal breakpoint boundaries were accurately defined. In total 15 breakpoints were identified.