Involvement of LFA-1 in lymphoma invasion and metastasis demonstrated with LFA-1-deficient mutants

Lymphocyte function-associated antigen-1 (LFA-1) is a leukocyte and lymphoma cell surface protein that promotes intercellular adhesion. We have previously shown that the invasion of hepatocyte cultures by lymphoma cells is inhibited by anti-LFA-1 antibodies (Roos, E., and F. F. Roossien. 1987. J. Cell Biol. 105:553-559). In addition, we now report that LFA-1 is also involved in invasion of lymphoma cells into fibroblast monolayers. To investigate the role of LFA-1 in metastasis of these lymphoma cells, we have generated mutants that are deficient in LFA-1 cell surface expression because of impaired synthesis of either the alpha or beta subunit precursor of LFA-1. We identified at least three distinct mutant clones. The invasive potential of the mutant cells in vitro, in both hepatocyte and fibroblast cultures, was considerably lower than that of parental cells. The metastatic potential of the mutants was much reduced, indicating that LFA-1 expression is required for efficient metastasis formation by certain lymphoma cells.     

Theoretical basis of protocols for seed storage

The protocols presently established for optimum seed storage do not account for the chemical composition of different seed species, the physiological status of the seed, and the physical status of water within the seed. The physiological status of seeds from five species with varying chemical compositions was determined by measurements of rates of oxygen uptake and seed deterioration. The physical status of water was determined by water sorption characteristics. For each species studied, there was a specific moisture content for the onset of respiration, chemical reactions, and accelerated aging rates. The moisture contents at which these physiological levels were observed varied among the species and correlated with the lipid content of the seed. However, the changes in physiological activities and the physical status of water occurred at specific relative humidities: 91% for the onset of respiration, 27% for the increased rates of thermal-chemical reactions, and 19% for optimum longevity. Based on these observations, we propose that equilibrating seeds between 19 and 27% relative humidity provides the optimum moisture level for maintaining seed longevity during longterm storage.     

Cloning and molecular analysis of the bifunctional dihydrofolate reductase-thymidylate synthase gene in the ciliated protozoanParamecium tetraurelia

We have cloned the first bifunctional gene dihydrofolate reductase-thymidylate synthase (DHFR-TS) from a free-living, ciliated protozoan,Paramecium tetraurelia, and determined its macronuclear sequence using a modified ligation-mediated polymerase chain reaction (PCR) that can be of general use in cloning strategies, especially where cDNA libraries are limiting. While bifunctional enzyme sequences are known from parasitic protozoa, none had previously been found in free-living protozoa. The AT-rich (68%) coding region spanning 1386 bp appears to lack introns. DHFR-TS localizes to a 500 kb macronuclear chromosome and is transcribed as an mRNA of 1.66 kb, predicted to encode a 53 kDa protein of 462 residues. The N-terminal one-third of the protein is encoded by DHFR, which is joined by a short junctional peptide of 12 amino acids to the highly conserved C-terminal TS domain. Among known DHFR-TS sequences, theP. tetraurelia gene is most similar to that fromToxoplasma gondii, based on primary sequence and parsimony analyses. The predicted secondary protein structure is similar to those of previously crystallized monofunctional sequences.     

Complete development of Cryptosporidium parvum in MDBK cells

Abstract Sporozoites of Cryptosporidium parvum excysted in vitro from bovine oocysts were incubated with monolayers of Madin-Darby bovine kidney cells. The extent of parasite colonisation was monitored by light microscopy and immunofluorescence. Electron microscopy confirmed the complete development and replication of C. parvum within Madin-Darby bovine kidney cells.     

The C-terminal KDEL sequence increases the expression level of a single-chain antibody designed to be targeted to both the cytosol and the secretory pathway in transgenic tobacco

The effects of subcellular localization on single-chain antibody (scFv) expression levels in transgenic tobacco was evaluated using an scFv construct of a model antibody possessing different targeting signals. For translocation into the secretory pathway a secretory signal sequence preceded the scFv gene (scFv-S). For cytosolic expression the scFv antibody gene lacked such a signal sequence (scFv-C). Also, both constructs were provided with the endoplasmic reticulum (ER) retention signal KDEL (scFv-SK and scFv-CK, respectively). The expression of the different scFv constructs in transgenic tobacco plants was controlled by a CaMV 35S promoter with double enhancer. The scFv-S and scFv-SK antibody genes reached expression levels of 0.01% and 1% of the total soluble protein, respectively. Surprisingly, scFv-CK transformants showed considerable expression of up to 0.2% whereas scFv-C transformants did not show any accumulation of the scFv antibody. The differences in protein expression levels could not be explained by the steady-state levels of the mRNAs. Transient expression assays with leaf protoplasts confirmed these expression levels observed in transgenic plants, although the expression level of the scFv-S construct was higher. Furthermore, these assays showed that both the secretory signal and the ER retention signal were recognized in the plant cells. The scFv-CK protein was located intracellularly, presumably in the cytosol. The increase in scFv protein stability in the presence of the KDEL retention signal is discussed.     

Formation of intracytoplasmic lipid inclusions by Rhodococcus opacus strain PD630

An oleaginous hydrocarbon-degrading Rhodococcus opacus strain (PD630) was isolated from a soil sample. The cells were able to grow on a variety of substrates and to produce large amounts of three different types of intracellular inclusions during growth on alkanes, phenylalkanes, or non-hydrocarbon substrates. Electron microscopy revealed large numbers of electron-transparent inclusions with a sphere-like structure. In addition, electron-dense inclusions representing polyphosphate and electron-transparent inclusions with an elongated disc-shaped morphology occurred in small amounts. The electron-transparent inclusions of alkane- or gluconate-grown cells were composed of neutral lipids (98%, w/w), phospholipids (1.2%, w/w), and protein (0.8%, w/w). The major component of the cellular inclusions was triacylglycerols; minor amounts of diacylglycerols and probably also some free fatty acids were also present. Free fatty acids and/or fatty acids in acylglycerols in cells of R. opacus amounted up to 76 or 87% of the cellular dry weight in gluconate- or olive-oil-grown cells, respectively. The fatty acid composition of the inclusions depended on the substrate used for cultivation. In cells cultivated on n-alkanes, the composition of the fatty acids was related to the substrate, and intermediates of the β-oxidation pathway, such as hexadecanoic or pentadecanoic acid, were among the acylglycerols. Hexadecanoic acid was also the major fatty acid (up 36% of total fatty acids) occurring in the lipid inclusions of gluconate-grown cells. This indicated that strain PD630 utilized β-oxidation and de novo fatty acid biosynthesis for the synthesis of storage lipids. Inclusions isolated from phenyldecane-grown cells contained mainly the non-modified substrate and phenylalkanoic acids derived from the hydrocarbon oxidation, such as phenyldecanoic acid, phenyloctanoic acid, and phenylhexanoic acid, and approximately 5% (w/w) of diacylglycerols. The lipid inclusions seemed to have definite structures, probably with membranes at their surfaces, which allow them to maintain their shape, and with some associated proteins, probably involved in the inclusion formation. Received: 22 December 1995 / Accepted: 12 March 1996     

Sporulation ofEnteromorpha spp. (Chlorophyta) and overwintering of spores in sediments of the Wadden Sea,Island Sylt,North Sea

For the last two decades dense mats of species of the filamentous green algaeEnteromorpha spp. have regulary occurred on tidal flats of Köningshafen Bay (island Sylt, North Sea, FRG). In calm areas overwintering of adult plants or plant fragments is a common process to guarantee the mass development during the next season. In contrast, the distribution ofEnteromorpha on exposed sandy tidal flats depends on recruitment by juvenile stages. In 1993Enteromorpha spore settlement was recorded regularly in the field. Polyethylene dishes were placed in the field and left for a period of seven days and lateron cultivated in the laboratory to checkEnteromorpha germling development. During summer 1993 — at a minimum distance of 200 m to the nearest adultEnteromorpha populations — a total of at least 82×10⁶ spores m^–2 settled. During winter the number of spores attached to the collecting dishes was close to zero and the adjacent sand flats were free of any visibleEnteromorpha plants. In further experiments it was shown that the development ofEnteromorpha juveniles in the next spring depended on the overwintering capacity of spores. More than 2×10⁶ spores m^–2 attached to large sand grains and other substrata (e.g. Hydrobia ulvae) survived the winter. In a laboratory experiment several species ofEnteromorpha were able to survive in total darkness for at least 10 months.