Exponential Megapriming PCR (EMP) Cloning—Seamless DNA Insertion into Any Target Plasmid without Sequence Constraints

We present a fast, reliable and inexpensive restriction-free cloning method for seamless DNA insertion into any plasmid without sequence limitation. Exponential megapriming PCR (EMP) cloning requires two consecutive PCR steps and can be carried out in one day. We show that EMP cloning has a higher efficiency than restriction-free (RF) cloning, especially for long inserts above 2.5 kb. EMP further enables simultaneous cloning of multiple inserts.     

Calculation of accurate small angle X-ray scattering curves from coarse-grained protein models

Background  

Genome sequencing projects have expanded the gap between the amount of known protein sequences and structures. The limitations of current high resolution structure determination methods make it unlikely that this gap will disappear in the near future. Small angle X-ray scattering (SAXS) is an established low resolution method for routinely determining the structure of proteins in solution. The purpose of this study is to develop a method for the efficient calculation of accurate SAXS curves from coarse-grained protein models. Such a method can for example be used to construct a likelihood function, which is paramount for structure determination based on statistical inference.     

Structure and immunochemistry of an oligosaccharide repeating unit of the capsular polysaccharide of type III group B Streptococcus. A revised structure for the type III group B streptococcal polysaccharide antigen

We have derived oligosaccharides from the capsular polysaccharide of type III group B Streptococcus by enzymatic hydrolysis of a specific backbone glycosidic bond utilizing an endo-beta-galactosidase from Flavobacterium keratolyticus. Enzymatic digestion of the polysaccharide produced oligosaccharide fragments of one or more pentasaccharide repeating units. On the basis of 13C NMR, 1H NMR, and methylation analyses, it was established that the smallest digestion fragment was alpha-D-NeupNAc-(2----3)-beta-D-Galp-(1----4)-[beta-D-Glcp-(1----6 )]- beta-D-GlcpNAc-(1----3)-beta-D-Gal. The isolation of this oligosaccharide is consistent with the susceptibility of the beta-D-Galp-(1----4)-beta-D-Glcp linkage in the backbone of the type III group B streptococcal polysaccharide and confirms that the polysaccharide is composed of a pentasaccharide repeating unit. High resolution 13C NMR spectroscopic studies indicated that, as in the case of the pentasaccharide, the terminal sialic acid residues of the type III group B streptococcal polysaccharide were linked to O-3 and not to O-6 of its branch beta-D-galactopyranosyl residues as had been previously reported (Jennings, H. J., Rosell, K.-G., and Kasper, D. L. (1980) Can. J. Chem. 58, 112-120). This linkage was confirmed in an independent methylation analysis of the type III group B streptococcal polysaccharide. Thin layer chromatogram binding assay and radioactive antigen binding assays with radiolabeled oligosaccharides demonstrated the single repeating unit pentasaccharide oligosaccharide to be poorly antigenic. Increasing oligosaccharide size to a decasaccharide consisting of two repeating units resulted in an 8-fold increase in antigen binding in the direct radioactive antigen binding assay. The results suggest that a region of the immunodeterminant site critical for antibody binding is located in the backbone of the polysaccharide and involves the beta-D-galactopyranose-(1----4) beta-D-glucopyranose bond.     

Immunohistochemical investigation of different cytokeratins and vimentin in the human epididymis from the fetal period up to adulthood

Summary The anatomical distribution of cytokeratins and vimentin was investigated by means of immunohistochemistry in the human epididymis. Epithelial cells of the ductuli efferentes and the corpus epididymidis were positive for cytokeratins and vimentin. The expression of epithelial vimentin decreased toward the cauda epididymidis, whereas cytokeratins remained unchanged. The epithelium of the ductus deferens was negative when antibodies against vimentin were used. With monoclonal antibodies to individual cytokeratins, the presence of cytokeratins 7, 8, 18, and 19 was demonstrated histochemically throughout the epithelium of the epididymis. Monoclonal antibodies specific for cytokeratin 17 allowed immunohistochemical differentiation between the ductuli efferentes and the ductus epididymidis.     

Further investigations on the clastogenicity of paracetamol and acetylsalicylic acid in vitro

Paracetamol (PCM) and acetylsalicylic acid (ASA), both widely used analgesics, were tested for their clastogenicity in V79 cells in vitro. Rat liver S9 mix and primary rat hepatocytes (PRH) were used as external activation systems. ASA was found to be negative with and without activation system in concentrations up to 10(-2) M. In contrast PCM induced concentration-dependent chromosomal aberrations with and without activation system within the range of 3 x 10(-3) and 10(-2) M. The greatest effects were observed following continuous treatment with PRH activation and without external metabolization. Pulse treatments without external metabolization, with S9 mix and PRH were less effective. The clastogenic potency of PCM seems to be partly independent of metabolic activation. Although clastogenic effects in vitro were observed only in very high concentrations pharmacokinetic data and other published mutagenicity data indicate that there might be a risk for human use. Peak plasma levels of more than 10(-4) M have been reported (Forrest et al., 1982) and 2 groups of investigators (Kocisova et al., 1988; Hongslo et al., 1990) found PCM to be weakly clastogenic in human lymphocytes in vivo in the maximum human therapeutic dose range.     

NADPH-cytochrome P-450 oxidoreductase: flavin mononucleotide and flavin adenine dinucleotide domains evolved from different flavoproteins

The FMN-binding domain of NADPH-cytochrome P-450 oxidoreductase, residues 77-228, is homologous with bacterial flavodoxins, while the FAD-binding domain, residues 267-678, shows a high degree of similarity to two FAD-containing proteins, ferredoxin-NADP+ reductase and NADH-cytochrome b5 reductase. Comparison of these proteins to glutathione reductase, a flavoprotein whose three-dimensional structure is known, has permitted tentative identification of FAD- and cofactor-binding residues in these proteins. The remarkable conservation of sequence between NADPH-cytochrome P-450 oxidoreductase and ferredoxin-NADP+ reductase, coupled with the homology of the FMN-binding domain of the oxidoreductase with the bacterial flavodoxins, implies that NADPH-cytochrome P-450 oxidoreductase arose as a result of fusion of the ancestral genes for these two functionally linked flavoproteins.     

Cellular control of abscess formation: role of T cells in the regulation of abscesses formed in response to Bacteroides fragilis

Although abscesses are a major sequela of infection, little is known about which cellular events initiate and which prevent this pathologic response. These studies are the first to indicate a role for T cells in the important pathogenic process of abscess development and also in immunity to abscesses induced by Bacteroides fragilis. We have shown that T cells initiate the formation of abscesses in mice after i.p. challenge with B. fragilis. These T cells bear both Ly-1 and Ly-2 surface markers. Nude mice (which have been shown by others to have T cell or T cell precursors) are also able to form abscesses. Cyclophosphamide-treated mice (with depressed T cell function) were not capable of developing abscesses. Reconstitution with normal or nude mouse spleen cells restored this ability. However, reconstitution with anti-Thy-1.2-treated, anti-Ly-1, or anti-Ly-2-treated spleen cells (or a mixture of the two cell populations) failed to allow abscess formation after bacterial challenge. Immunity to abscesses caused by B. fragilis requires two T cells. The first Ly-1-2+ T cell has an IJ surface marker and has been shown to release a small m.w. soluble factor (ITF) that is antigen specific. Immunity to abscesses, however, depends on the interaction of ITF with a second Ly-1-2+ T cell, demonstrated in reconstitution experiments with nude mice. The data presented document a critical role for T cells in abscess induction and suggest the existence of a suppressor-like T cell circuit in immunity to abscesses.     

G2 arrest and impaired nucleocytoplasmic transport in mouse embryos lacking the proto-oncogene CAN/Nup214.

The vertebrate nucleopore complex (NPC) is a 125 MDa multiprotein assembly that mediates nucleocytoplasmic transport. One of its components, CAN/Nup214, is an FXFG repeat-containing protein known to be involved in myeloid leukemia in humans. We have devised a powerful genetic approach, using maternally derived protein in murine null embryos, to show that CAN/ Nup214 is essential for NPC function in vivo. We demonstrate that CAN-/- mouse embryonic stem (ES) cells are not viable and that CAN-/- embryos die in utero between 4.0 and 4.5 days postcoitum, following the depletion of their CAN from maternal sources. In 3.5-day-old mutant embryos, cultured in vitro, progressive depletion of CAN leads to cell cycle arrest in G2 phase, and eventually to blastocoel collapse, impaired NLS-mediated protein uptake and nuclear accumulation of polyadenylated RNA. Remarkably, these defective CAN-depleted embryos do not display any gross morphological abnormalities in their nuclear envelopes or NPCs. Our data suggest that CAN is critical to cell cycle progression and required for both nuclear protein import and mRNA export.     

Hopped Beer: The Case For Cultivation

The history of hops, hopped beer, and hop cultivation is unclear and ambiguous. An assessment of the available literature reveals many contradictions, especially regarding the first use of hops in beer and the earliest incidence of hop cultivation. Historically, hops were used for a variety of purposes; now their primary use is as a preservative and flavoring in beer. Hop cultivation is poorly documented, but was certainly undertaken by the 10th century, most probably in response to the demand generated by beer-brewing. After comparing the literature and investigating source material, a chronology of hop use in beer and hop cultivation is proposed.