Evaluation of mass spectrometric techniques for charaterization of engineered proteins

A simple and versatile method of in vitro site-specific mutagenesis based on polymerase chain reaction (PCR) is described. The complete method required the use of three oligonucleotide primers and two PCRs. The product of the first PCR was used as one of the primers (megaprimer) in the second PCR. Essentially 100% of the final product incorporated the desired mutation. The various aspects of the procedure and its application is described in detail.     

Mytilid bivalveLithophaga in Upper Triassic coralPamiroseris from Zlambach beds compared with cretaceousLithophaga Alpina

Summary The mytilid genusLithophaga is confirmed for the Upper Triassic. The Rhaetian specimen, boring the dead part of coral, is compared with the SenonianL. alpina, which is associated with live coral.     

Magnetic-field flux density and spectral characteristics of motor-driven personal appliances

Flux density and spectral measurements were carried out on magnetic fields generated by several types of motor-driven personal appliances used near the body. Among the units tested were several for which the average flux densities, as determined at the surfaces of the appliance, exceeded 0.4 mT. Time-rates-of-change (dB/dt) for several units exceeded 1000 T/s, and several units exhibited high-frequency components in the low-MHz range. Use of such appliances, although normally of short duration, can represent exposure to magnetic fields of relatively high flux density, which may also have high-frequency components. Compared to other household and commercial sources of magnetic fields, those generated by certain motor-driven personal appliances may represent a significant contribution to time-weighted average exposure and may represent an important source of local induced currents in the body. Furthermore, high-frequency transients that represent only a minor contribution to time-weighted average exposure may generate significant instantaneous induced currents. © 1994 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Inheritance of Strain Instability (Sectoring) in the Commercial Button Mushroom, Agaricus bisporus

The button mushroom, Agaricus bisporus, is a commercially important cultivated filamentous fungus. During the last decade, the button mushroom industry has depended mainly on two strains (or derivatives of these two strains). Using one of these highly successful strains (strain U1) we examined the phenomenon of strain instability, specifically, the production of irreversible sectors. Three “stromatal” and three “fluffy” sectors were compared with a healthy type U1 strain and with a wild-collected isolate. Compost colonization and fruit body morphology were examined. The main objective of this study, however, was to examine the meiotic stability of the sectored phenotype. Single basidiospores were isolated and subjected to a grain bioassay in which the ability to produce sectors was measured. Our results were as follows: (i) basidiospore cultures obtained from a wild-collected isolate showed no tendency to produce sectors; (ii) approximately 5% of the basidiospore cultures obtained from healthy type U1 strains produced irreversible sectors in the grain bioassay; (iii) the five primary sectors examined produced basidiospore cultures, half of which produced normal-looking growth in the grain bioassay and half of which produced some degree of sectoring; and (iv) the one sectored isolate that represented the F2 generation gave ratios similar to the 1:1 ratio observed for the F1 cultures.     

The effects of nutrient addition and pH manipulation in bag experiments on the phytoplankton of a small acidic lake in West Virginia,USA

In situ bag experiments were performed during summer and autumn in a small acidic lake, Tibbs Run Lake, West Virginia, USA. The objective was to evaluate phytoplankton responses to pH manipulation and nutrient addition. Increasing the pH from below 4.5 to over 6.3 resulted in great declines in phytoplankton biovolume. There was also a succession from dinoflagellates (Peridinium inconspicuum to small chlorophytes. The trend was more rapid where phosphorus (P) additions were made along with pH enhancement. During summer, P limitation was indicated, while nitrogen (N) appeared to limit production in autumn. In both seasons, nutrient additions greatly altered the phytoplankton composition in high pH treatments, but had no discernable effects at (the natural) low pH. A low pH, P addition treatment in autumn was the single exception. When N was subsequently added, phytoplankton composition changed dramatically, probably because the proceeding P additions caused severe secondary N-limitation. In general, however, the results supported the view that phytoplankton compositional responses to nutrient additions are suppressed in low pH, relative to high pH lake water.     

HIV-1 integrase blocks infection of bacteria by single-stranded DNA and RNA bacteriophages

Expression of human immunodeficiency virus-1 integrase in Escherichia coli, at levels that had no effect on bacterial cell growth, blocked plaque formation by bacteriophages having single-stranded genomic DNA (M13) or RNA (R17, Q, PRR1). Plaque formation by phages having double-stranded genomic DNA (T4, PR4) was unaffected. Integrase also inhibited infection by the phagemid M13KO7, but it had no effect on production of phage once infection by M13KO7 was established. This result indicated that integrase affects an early stage in infection. Integrase also inhibited phage production following transfection by either single-stranded or double-stranded (replicative form) M13 DNA, it blocked M13 DNA replication, as assayed by incorporation of radioactive nucleotides into DNA, and it failed to affect bacterial pilus function. These data suggest that integrase interacts in vivo with phage nucleic acid, a conclusion supported by studies in which integrase was shown to have a DNA-binding activity in its C-terminal portion. This portion of integrase was both necessary and sufficient for interference of plaque formation by M13 in the present study. Expression of the N-terminal portion of integrase at the same level as intact integrase had little effect on phage growth, indicating that expression of foreign protein in general was not responsible for the inhibitory effect. The simple bacteriophage assay described is potentially useful for identifying integrase mutants that lack single-stranded DNA binding activity.     

Structural analysis and molybdenum-dependent expression of the pAO1-encoded nicotine dehydrogenase genes of Arthrobacter nicotinovorans

The genes of nicotine dehydrogenase (NDH) were identified, cloned and sequenced from the catabolic plasmid pA01 of Arthrobacter nicotinovorans. In immediate proximity to this gene cluster is the beginning of the 6-hydroxy-L-niotine oxidase (6-HLNO) gene. NDH is composed of three subunits (A, B and C) of M_r 30011, 14924 and 87677. It belongs to a family of bacterial hydroxylases with a similar subunit structure; they have molybdopterin dinucleotide, FAD and Fe-S clusters as cofactors. Here the first complete primary structure of a bacterial hydroxylase is provided. Sequence alignments of each of the NDH subunits show similarities to the sequences of eukaryotic xanthine dehydrogenase (XDH) but not to other known molybdenum-containing bacterial enzymes. Based on alignment with XDH it is inferred that the smallest subunit (NDHB) carries an iron-sulphur cluster, that the middle-sized subunit (NDHA) binds FAD, and that the largest NDH subunit (NDHC) corresponds to the molybdopterin-binding domain of XDH. Expression of both the ndh and the 6-hlno genes required the presence of nicotine and molybdenum in the culture medium. Tungsten inhibited enzyme activity but not the synthesis of the enzyme protein. The enzyme was found in A. nicotinovorans cells in a soluble form and in a membrane-associated form. In the presence of tungsten the fraction of membrane-associated NDH increased.     

Carbohydrate receptor-mediated gene transfer to human T leukaemic cells

The mucin-type carbohydrate Tn cryptantigen (GalNAc1-O-Ser/Thr,where GalNAc is N-acetyl-D-galactosamine) is expressed in manycarcinomas, in haemopoietic disorders including the Tn syndrome,and on human immunodeficiency virus (HIV) coat glycoproteins,but is not expressed on normal, differentiated cells becauseof the expression of a Tn-processing galactosyltransferase.Using Jurkat T leukaemic cells which express high levels ofTn antigen due to deficient Tn galactosylation, we have establishedthe Tn antigen-mediated gene transfer and demonstrate the considerableefficiency of this approach. We used poly(L-lysine) conjugatesof the monoclonal antibody 1E3 directed against the Tn antigento deliver the luciferase and ß-galactosidase reportergenes to Jurkat cells by receptor-mediated endocytosis. Additionof unconjugated 1E3 reduced transfection efficiency in a concentration-dependentmanner and incubation with free GalNAc abolished DNA transfercompletely, indicating that gene delivery is indeed mediatedby the Tn antigen. Pre-treatment of Jurkat cells with Vibriocholerae sialidase, which uncovers additional Tn antigens, resultedin an improvement of gene transfection. Both human and chickenadenovirus particles attached to the DNA/polylysine complexstrongly augmented transgene expression. When the ß-galactosidase(lacZ) gene was delivered to Jurkat cells by Tn-mediated endocytosis,up to 60% of the cells were positive in the cytochemical stainusing 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside(X-gal) as a chromogenic substrate. The efficiency of the transferrinreceptor-mediated DNA uptake into Jurkat cells was comparativelylow, although these cells were shown to express considerableamounts of transferrin receptor. We show here that a mucin-typecarbohydrate antigen mediates highly efficient DNA uptake byendocytosis into Jurkat T cells. This method represents a 50-foldimprovement of Jurkat cell transfection efficiency over otherphysical gene transfer techniques. Specific gene delivery toprimary cancer cells exhibiting Tn epitopes may especially bedesirable in immunotherapy protocols. adenovirus endocytosis gene transfer T cell Tn antigen     

Expression of CD44 Splice Variants During Lymphocyte Activation and Tumor Progression

Recently, splice variants of CD44 have been described that confer metastatic potential to non-metastasizing rat pancreatic carcinoma and sarcoma cell lines. Using antibodies against variant CD44 (CD44v) sequences, we have examined the expression of variant CD44 glycoproteins on human lymphoid cells and tissues and in colorectal neoplasia. Lymphohematopoietic cells express low levels of CD44v glycoproteins. During the process of lymphocyte activation in vitro and in vivo, expression of CD44v glycoproteins is transiently upregulated. The reaction pattern of various antibodies indicates that these CD44 variants contain the domain encoded by exon v6, which is part of the variant that confers metastatic capability. In human colorectal neoplasia we observed overexpression of CD44 splice variants in all invasive carcinomas. Already at early stages of colorectal tumor progression exon v5 epitopes were overexpressed. Tumor progression was strongly related to expression of CD44 isoforms containing exon v6 encoded domains. The findings establish CD44 variants as tumor progression markers in colorectal cancer.     

Evidence for the Colonization of Lactic Acid Bacteria in the Gastrointestinal Tract of Suckling Mink

Lactic acid bacteria are considered indigenous members of the gastrointestinal microflora in a number of animal species (Savage 1977a). Some intestinal strains of lactobacilli and streptococci are aWe to adhere to stratified squamous epithelium of some animals (Tannock et al. 1987), in the non-secreting part of the stomach of piglets (Barrow et al. 1980, Fuller et al. 1978) and rodents (Tannock et al. 1982), and in the crop of poultry (Fuller 1978). The presence of lactic acid bacteria in the digestive tract is believed to be of beneficial value to the host animal (Fuller 1989). The production of organic acids in the stomach or the crop helps maintaining a low pH which may be important for inhibiting the colonization of potentially pathogenic bacteria, particularly in the newborn animal (Barrow et al 1980, Fuller 1977, Fuller 1978). The adhesion of lactobacilli to squamous epithelium is host specific: strains capable of adhering to the epithelium of piglets are usually not able to adhere in rodents or poultry and vice versa (Fuller 1978, Lin & Savage 1984, Tannock et al 1982). Adhesion of lactic acid bacterial strains to other epithelia than stratified squamous epithelium has been reported. Thus, the attachment of lactobacilli to cells from the secreting epithelium of the murine stomach (Kotarski & Savage 1979), to intestinal cells of humans (Goldin & Gorbach 1987), and to columnar epithelial cells of piglets and calves (Mäyrä-Mäkinen et al 1983) has been demonstrated using in vitro methods. In another study the in vivo attachment of Enterococcus faecium to duodenal epithelium of gnotobi-otic chickens was demonstrated (Fuller et al 1981). Recent research indicated that in adult mink lactic acid bacteria are not indigenous members of the intestinal flora, and they do not attach to epithelium in any part of the gastrointestinal tract (Federsen & Jørgensen 1992). The present paper presents evidence that Gram positive cocci may colonize the gut of suckling mink kits and attach to the gut mucosa.